Human antibodies to GREM1

ABSTRACT

The present invention provides antibodies that bind to human gremlin-1 (GREM1), and methods of use. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to GREM1. The antibodies of the invention are useful for inhibiting or neutralizing GREM1 activity, thus providing a means of treating a GREM1-related disease or disorder such as fibrosis and cancer. In some embodiments, the antibodies of the present invention are used in treating at least one symptom or complication of fibrosis of the liver, lungs or kidney.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/775,637,filed Sep. 11, 2015, which is a US national stage of InternationalApplication No. PCT/US2014/021471, filed Mar. 7, 2014, which claims thebenefit under 35 USC § 119(e) of U.S. Provisional Application No.61/782,874, filed Mar. 14, 2013, and 61/883,218, filed Sep. 27, 2013.Each of these applications is incorporated herein by reference in itsentirety for all purposes.

SEQUENCE LISTING

This application incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file 9800US02-Sequence.txt,created on Jun. 27, 2019 and containing 196,031 bytes.

FIELD OF THE INVENTION

The present invention is related to human antibodies and antigen-bindingfragments of human antibodies that specifically bind to human gremlin-1(GREM1), and therapeutic and diagnostic methods of using thoseantibodies.

STATEMENT OF RELATED ART

Fibrosis is a scarring process that is a common feature of chronic organinjury. It is characterized by elevated activity of transforming growthfactor-beta (TGF-β) resulting in increased and altered deposition ofextracellular matrix and other fibrosis-associated proteins.

Bone morphogenetic proteins (BMPs) are phylogenetically conservedsignaling molecules that belong to the TGF-β superfamily and areinvolved in growth, development and differentiation of various celltypes (Yanagita, M., 2009, Biofactors DOI: 10:1002/biof.15). Thebiological responses to BMPs are negatively regulated by BMP antagoniststhat can directly associate with BMPs and inhibit receptor binding.Human gremlin-1 (GREM1), a member of the cysteine knot superfamily, isan antagonist for BMP signaling (Hsu, D. R., et al 1998, Mol. Cell 1:673-683). It binds to BMP2, BMP4 and BMP7. GREM1 blocks BMP signaling,which is thought to be anti-fibrotic in many tissues by blocking thebinding of BMP to its receptor.

The expression of GREM1 in normal adult kidney, liver and lung is verylow. However, GREM1 expression is increased in both mouse models offibrosis and human fibrotic diseases such as diabetic nephropathy andpulmonary fibrosis (Koli et al., 2006, Am. J. Pathol. 169: 61-71;Farkas, et al., 2011, Am. J. Respir. Cell. Mol. Biol. 44: 870-878;Lappin, et al., 2002, Nephrol. Dial. Transplant. 17: 65-67). IncreasedGREM1 expression leads to a reduction in anti-fibrotic BMP signaling.Increased GREM1 expression also correlates with increased serumcreatinine levels and tubulointerstitial fibrosis scores in thesediseases (Dolan, V., et al 2005, Am. J. Kidney Dis. 45: 1034-9). Inseveral fibrosis models, such as lung and kidney fibrosis, theexpression of GREM1 is greatly increased while BMP signaling isdecreased (Myllarniemi, et al., 2008, Am. J. Respir. Crit. Care Med.177: 321-329). Administration of BMP7 can decrease fibrosis in somemodels of kidney disease, but does not protect against bleomycin-inducedlung or skin fibrosis (Weiskirchen, et al., 2009, 14: 4992-5012).

Mice heterozygous for GREM1 show some protection against fibrosis in anexperimental model of diabetic nephropathy (Zhang, et al., 2009, BBRC383: 1-3). These mice show no difference in the onset, severity andprogression of diabetes as measured by weight loss and hyperglycemia.They do, however, have attenuated fibrotic structural changes in thekidney and reduced changes in kidney function.

GREM1 may thus serve as a potential therapeutic target for the treatmentof fibrotic diseases. There is a need to develop specific GREM1inhibitors in fibrosis treatment which do not have any side-effects.

In addition, GREM1 is an agonist of the major proangiogenic receptorvascular endothelial growth factor receptor-2 (VEGFR-2) and may play anoncogenic role especially in carcinomas of the uterine cervix, lung,ovary, kidney, breast, colon, pancreas, and sarcoma (Namkoong et al2006, BMC Cancer 6: 74 doi:10.1186/1471-2407-6-74; Mitola et al 2010;Blood 116: 3677-3680). Heparan sulfate (HS) and heparin,glycosaminoglycans (GAGs) known for their anticoagulant effects, havebeen shown to bind to GREM1. GREM1 binds to heparin and activatesVEGFR-2 in a BMP-independent manner (Chiodelli et al 2011; Arterioscler.Thromb. Vasc. Biol. 31: e116-e127).

Anti-GREM1 polyclonal and monoclonal antibodies are availablecommercially (for example, from Sigma-Aldrich, Abnova Corporation, NovusBiologicals, Genway). U.S. Pat. No. 6,432,410 discloses the nucleotideand protein sequences of human, mouse, xenopus and chick GREM1 anddeletion mutants thereof. US20090203041 discloses GREM1 peptidesequences for use as BMP4 inhibitors. Kim et al disclose GREM1antibodies which inhibit GREM1 in a manner independent of BMP or VEGFR-2in PLoS One 7(4): e35100. doi:10.1371/journal.pone.0035100 and inWO2013137686. U.S. Pat. No. 7,744,873 discloses methods for treatingglaucoma by administering a GREM1 antagonist, wherein the antagonist isan antibody that binds GREM1. Methods of treatment and formulations forglaucoma or cancer using GREM1 antagonists including antibodies havebeen described in EP1440159B1, EP1777519A1, EP2053135A1, and US20090041757.

BRIEF SUMMARY OF THE INVENTION

The invention provides fully human monoclonal antibodies (mAbs) andantigen-binding fragments thereof that bind specifically to human GREM1.Such antibodies may be useful to neutralize the activity of GREM1 or toblock binding of GREM1 to a bone morphogenetic protein (BMP) such asBMP2, BMP4 or BMP7. In certain other embodiments, the antibodies may beuseful to neutralize the activity or block binding to heparin or heparansulfate. The antibodies may act to halt the progression, or to lessenthe severity of a fibrosis-associated condition or disease, or reducethe number, the duration, or the severity of disease recurrence, orameliorate at least one symptom associated with fibrosis or cancer. Suchantibodies may be used alone or in conjunction with a second agentuseful for treating fibrosis or cancer. In certain embodiments, theantibodies specific for GREM1, may be given therapeutically inconjunction with a second agent to lessen the severity of thefibrosis-associated condition or cancer, or to reduce the number, theduration, or the severity of disease recurrence, or ameliorate at leastone symptom associated with the fibrosis-associated condition or cancer.In certain embodiments, the antibodies may be used prophylactically asstand-alone therapy to protect patients who are at risk for developing afibrosis-associated condition or disease. For example, certain patientpopulations may be at risk for developing a fibrosis condition ordisease, including elderly patients, or patients with family history, orpatients with problems of alcohol or drug abuse, or patients who havechronic and/or concomitant underlying medical conditions such asdiabetes, metabolic disorders, liver injury, renal injury or lung injurythat may pre-dispose them to fibrosis. Other at-risk patient populationsinclude individuals exposed to chemicals such as asbestos or otherpollutants, or smokers. Any of these patient populations may benefitfrom treatment with the antibodies of the invention, when given alone orin conjunction with a second agent.

The antibodies of the present invention may be used to treat fibrosis inlungs, liver, kidney, skin, heart, gut or muscle of a patient. In otherembodiments, the antibodies of the invention may be used to treat cancersuch as carcinoma of the uterine cervix, lung, ovary, kidney, breast,colon, or pancreas. The antibodies can be full-length (for example, anIgG1 or IgG4 antibody) or may comprise only an antigen-binding portion(for example, a Fab, F(ab′)₂ or scFv fragment), and may be modified toaffect functionality, e.g., to eliminate residual effector functions(Reddy et al., (2000), J. Immunol. 164:1925-1933).

Accordingly, in a first aspect, the invention provides an isolated fullyhuman monoclonal antibody or antigen-binding fragment thereof that bindsto human GREM1.

In one embodiment, the human monoclonal antibody binds to GREM1 of SEQID NO: 594 or SEQ ID NO: 595.

In one embodiment, the isolated antibody or antigen-binding fragmentthereof blocks GREM1 binding to BMP2, BMP4, BMP7 or heparin.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof binds to GREM1 with a K_(D) equal to or less than 10⁻⁷M as measured by surface plasmon resonance.

In one embodiment, the isolated antibody or antigen-binding fragmentthereof exhibits one or more properties selected from the groupconsisting of: (a) binds GREM1 at 37° C. with a binding dissociationequilibrium constant (K_(D)) of less than about 275 nM as measured bysurface plasmon resonance; (b) binds to GREM1 at 37° C. with adissociative half-life (t½) of greater than about 3 minutes as measuredby surface plasmon resonance; (c) binds GREM1 at 25° C. with a K_(D) ofless than about 280 nM as measured by surface plasmon resonance; (d)binds to GREM1 at 25° C. with a t½ of greater than about 2 minutes asmeasured by surface plasmon resonance; (e) blocks GREM1 binding to BMP4with an IC₅₀ of less than about 1.9 nM as measured in a competitionELISA assay at 25° C.; (f) blocks GREM1-mediated inhibition of BMPsignaling and promotes cell differentiation; and (g) blocks GREM1binding to heparin.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to GREM1 comprises three heavy chaincomplementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3)contained within any one of the heavy chain variable region (HCVR)sequences selected from the group consisting of SEQ ID NOs: 2, 18, 34,50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274,290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482, 498,514, 530, 546, 562, and 578; and three light chain CDRs (LCDR1, LCDR2and LCDR3) contained within any one of the light chain variable region(LCVR) sequences selected from the group consisting of SEQ ID NOs: 10,26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250,266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474,490, 506, 522, 538, 554, 570, and 586. Methods and techniques foridentifying CDRs within HCVR and LCVR amino acid sequences are wellknown in the art and can be used to identify CDRs within the specifiedheavy chain variable region(s) (HCVR) and/or light chain variableregion(s) (LCVR) amino acid sequences disclosed herein. Exemplaryconventions that can be used to identify the boundaries of CDRs include,e.g., the Kabat definition, the Chothia definition, and the AbMdefinition. In general terms, the Kabat definition is based on sequencevariability, the Chothia definition is based on the location of thestructural loop regions, and the AbM definition is a compromise betweenthe Kabat and Chothia approaches. See, e.g., Kabat, “Sequences ofProteins of Immunological Interest,” National Institutes of Health,Bethesda, Md. (1991); Al-Lazikani et al., (1997), J. Mol. Biol.273:927-948; and Martin et al., (1989), Proc. Natl. Acad. Sci. USA86:9268-9272. Public databases are also available for identifying CDRsequences within an antibody.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to GREM1 comprises a HCVR having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 18,34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258,274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482,498, 514, 530, 546, 562, and 578.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to GREM1 comprises a LCVR having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 10, 26,42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266,282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490,506, 522, 538, 554, 570 and 586.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to GREM1 comprises (a) a HCVR having anamino acid sequence selected from the group consisting of SEQ ID NOs: 2,18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242,258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466,482, 498, 514, 530, 546, 562, and 578; and (b) a LCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO: 10, 26,42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266,282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490,506, 522, 538, 554, 570 and 586.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to GREM1 comprises:

-   (a) a HCDR1 domain having an amino acid sequence selected from the    group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116,    132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324,    340, 356, 372, 388, 404, 420, 436, 452, 468, 484, 500, 516, 532,    548, 564, and 580;-   (b) a HCDR2 domain having an amino acid sequence selected from the    group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118,    134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326,    342, 358, 374, 390, 406, 422, 438, 454, 470, 486, 502, 518, 534,    550, 566 and 582;-   (c) a HCDR3 domain having an amino acid sequence selected from the    group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120,    136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328,    344, 360, 376, 392, 408, 424, 440, 456, 472, 488, 504, 520, 536,    552, 568 and 584;-   (d) a LCDR1 domain having an amino acid sequence selected from the    group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124,    140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332,    348, 364, 380, 396, 412, 428, 444, 460, 476, 492, 508, 524, 540,    556, 572 and 588;-   (e) a LCDR2 domain having an amino acid sequence selected from the    group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126,    142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334,    350, 366, 382, 398, 414, 430, 446, 462, 478, 494, 510, 526, 542,    558, 574, and 590; and-   (f) a LCDR3 domain having an amino acid sequence selected from the    group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128,    144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336,    352, 368, 384, 400, 416, 432, 448, 464, 480, 496, 512, 528, 544,    560, 576 and 592.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to GREM1 comprises a HCVR/LCVR amino acidsequence pair selected from the group consisting of SEQ ID NOs: 2/10,18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154,162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282,290/298, 306/314, 322/330, 338/346, 354/362, 370/378, 386/394, 402/410,418/426, 434/442, 450/458, 466/474, 482/490, 498/506, 514/52, 530/538,546/554, 562/570, and 578/586.

In one embodiment, the invention provides a fully human monoclonalantibody or antigen-binding fragment thereof that binds to GREM1,wherein the antibody or fragment thereof exhibits one or more of thefollowing characteristics: (i) comprises a HCVR having an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50,66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290,306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482, 498, 514,530, 546, 562, and 578, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; (ii) comprises a LCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106,122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330,346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554,570, and 586, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;(iii) comprises a HCDR3 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120,136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344,360, 376, 392, 408, 424, 440, 456, 472, 488, 504, 520, 536, 552, 568,and 584, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; and aLCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160,176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384,400, 416, 432, 448, 464, 480, 496, 512, 528, 544, 560, 576, and 592, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; (iv) comprises aHCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164,180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388,404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, and 580, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a HCDR2 domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214,230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438,454, 470, 486, 502, 518, 534, 550, 566, and 582, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; a LCDR1 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 12, 28, 44,60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284,300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476, 492, 508,524, 540, 556, 572, and 588, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; and a LCDR2 domain having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126,142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350,366, 382, 398, 414, 430, 446, 462, 478, 494, 510, 526, 542, 558, 574,and 590, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; (v)binds to GREM1 with a K_(D) equal to or less than 10⁻⁷ M as measured bysurface plasmon resonance.

In a second aspect, the invention provides an isolated antibody orantigen-binding fragment thereof that competes for specific binding tohuman GREM1 with an antibody or antigen-binding fragment comprising thecomplementarity determining regions (CDRs) of a heavy chain variableregion (HCVR), wherein the HCVR has an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130,146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354,370, 386, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546, 562, and578; and the CDRs of a light chain variable region (LCVR), wherein theLCVR has an amino acid sequence selected from the group consisting ofSEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202,218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426,442, 458, 474, 490, 506, 522, 538, 554, 570, and 586.

In one embodiment, the invention provides an isolated antibody orantigen-binding fragment thereof that binds the same epitope on humanGREM1 as an antibody or antigen-binding fragment comprising the CDRs ofa heavy chain variable region (HCVR), wherein the HCVR has an amino acidsequence selected from the group consisting of SEQ ID NOs: 2, 18, 34,50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274,290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482, 498,514, 530, 546, 562, and 578; and the CDRs of a light chain variableregion (LCVR), wherein the LCVR has an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122,138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346,362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554, 570,and 586.

In one embodiment, the invention provides for an isolated antibody orantigen-binding fragment thereof that blocks binding of human GREM1 toany one of BMP2, BMP4, BMP7 or heparin, the antibody comprising thecomplementarity determining regions (CDRs) of a heavy chain variableregion (HCVR), wherein the HCVR has an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130,146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354,370, 386, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546, 562, and578; and the CDRs of a light chain variable region (LCVR), wherein theLCVR has an amino acid sequence selected from the group consisting ofSEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202,218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426,442, 458, 474, 490, 506, 522, 538, 554, 570, and 586.

In one embodiment, the invention provides a fully human monoclonalantibody or antigen-binding fragment thereof that binds to GREM1,wherein the antibody or fragment thereof exhibits one or more of thefollowing characteristics: (i) comprises a HCVR having an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50,66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290,306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482, 498, 514,530, 546, 562, and 578, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; (ii) comprises a LCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106,122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330,346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554,570, and 586, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;(iii) comprises a HCDR3 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120,136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344,360, 376, 392, 408, 424, 440, 456, 472, 488, 504, 520, 536, 552, 568,and 584, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; and aLCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160,176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384,400, 416, 432, 448, 464, 480, 496, 512, 528, 544, 560, 576, and 592, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; (iv) comprises aHCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164,180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388,404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, and 580, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a HCDR2 domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214,230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438,454, 470, 486, 502, 518, 534, 550, 566, and 582, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; a LCDR1 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 12, 28, 44,60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284,300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476, 492, 508,524, 540, 556, 572, and 588, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; and a LCDR2 domain having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126,142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350,366, 382, 398, 414, 430, 446, 462, 478, 494, 510, 526, 542, 558, 574,and 590, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; (v)binds to GREM1 with a K_(D) equal to or less than 10⁻⁷ M as measured bysurface plasmon resonance; (vi) blocks GREM1 binding to one of BMP2,BMP4 or BMP7; (vii) blocks GREM1-inhibition of BMP signaling andpromotes cell differentiation; and (viii) blocks GREM1 binding toheparin.

In a third aspect, the invention provides nucleic acid moleculesencoding anti-GREM1 antibodies or fragments thereof. Recombinantexpression vectors carrying the nucleic acids of the invention, and hostcells into which such vectors have been introduced, are also encompassedby the invention, as are methods of producing the antibodies byculturing the host cells under conditions permitting production of theantibodies, and recovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113,129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337,353, 369, 385, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561,and 577, or a substantially identical sequence having at least 90%, atleast 95%, at least 98%, or at least 99% homology thereof.

In one embodiment, the antibody or fragment thereof further comprises aLCVR encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169,185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361, 377, 393,409, 425, 441, 457, 473, 489, 505, 521, 537, 553, 569, and 585, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

In one embodiment, the invention provides an antibody or antigen-bindingfragment of an antibody comprising a HCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 7,23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247,263, 279, 295, 311, 327, 343, 359, 375, 391, 407, 423, 439, 455, 471,487, 503, 519, 535, 551, 567, and 583, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; and a LCDR3 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 15, 31, 47,63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287,303, 319, 335, 351, 367, 383, 399, 415, 431, 447, 463, 479, 495, 511,527, 543, 559, 575, and 591, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In one embodiment, the invention provides an antibody or fragmentthereof further comprising a HCDR1 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 3, 19, 35, 51,67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291,307, 323, 339, 355, 371, 387, 403, 419, 435, 451, 467, 483, 499, 515,531, 547, 563, and 579, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; a HCDR2 domain encoded by a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117, 133,149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309, 325, 341, 357,373, 389, 405, 421, 437, 453, 469, 485, 501, 517, 533, 549, 565, and581, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a LCDR1domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155,171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379,395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555, 571, and 587, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a LCDR2 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205,221, 237, 253, 269, 285, 301, 317, 333, 349, 365, 381, 397, 413, 429,445, 461, 477, 493, 509, 525, 541, 557, 573, and 589, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

In some embodiments, the antibody or antigen-binding fragment thereof tohuman GREM1, as described herein may be linked to a detectable labelsuch as a radionuclide label or a MRI-detectable label.

In a fourth aspect, the invention provides a pharmaceutical compositioncomprising an isolated fully human monoclonal antibody orantigen-binding fragment thereof that binds to GREM1 and apharmaceutically acceptable carrier or diluent. In one embodiment, theinvention provides a pharmaceutical composition comprising an isolatedfully human monoclonal antibody or antigen-binding fragment thereof thatbinds specifically to the secreted form of human GREM1 and apharmaceutically acceptable carrier or diluent. In one embodiment, theinvention provides a pharmaceutical composition comprising an isolatedfully human monoclonal antibody or antigen-binding fragment thereof thatbinds specifically to the membrane-associated form of GREM1 (matureGREM1 protein) and a pharmaceutically acceptable carrier or diluent.

In one embodiment, the pharmaceutical composition comprises a fullyhuman monoclonal antibody that binds to GREM1 having any one or more ofthe characteristics described herein. The antibody that binds to GREM1binds with a K_(D) equal to or less than 10⁻⁷M.

In one embodiment, the composition comprises an antibody that binds tohuman GREM1 and has a HCVR/LCVR amino acid sequence pair selected fromthe group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74,82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202,210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 322/330,338/346, 354/362, 370/378, 386/394, 402/410, 418/426, 434/442, 450/458,466/474, 482/490, 498/506, 514/522, 530/538, 546/554, 562/570, and578/586.

In one embodiment, the invention features a composition, which is acombination of an antibody or antigen-binding fragment of an antibody ofthe invention, and a second therapeutic agent.

The second therapeutic agent may be a small molecule drug, aprotein/polypeptide, an antibody, a nucleic acid molecule, such as ananti-sense oligonucleotide, or a siRNA. The second therapeutic agent maybe synthetic or naturally derived.

The second therapeutic agent may be any agent that is advantageouslycombined with the antibody or fragment thereof of the invention, forexample, an anti-fibrotic drug such as pirfenidone, an antibiotic, ananti-inflammatory drug, a non-steroidal anti-inflammatory drug (NSAID),a cytotoxic agent, a chemotherapeutic agent, a corticosteroid such asprednisone, an endothelin receptor antagonist such as Bosentan,macitentan or ambrisentan, a nutritional supplement, ananti-hypertensive agent, an antioxidant, a vascular endothelial growthfactor (VEGF) antagonist [e.g., a “VEGF-Trap” such as aflibercept orother VEGF-inhibiting fusion protein as set forth in U.S. Pat. No.7,087,411, or an anti-VEGF antibody or antigen binding fragment thereof(e.g., bevacizumab, or ranibizumab)], another antibody that binds toGREM1, or an antibody against a chemokine such as TGF-β, or against ancytokine such as IL-1, anti-LOXL2, anti-avb6integrin, a galectin-3targeting drug, imatinib or any other PDGFR antagonist and anti-AOC3drugs.

In certain embodiments, the second therapeutic agent may be an agentthat helps to counteract or reduce any possible side effect(s)associated with the antibody or antigen-binding fragment of an antibodyof the invention, if such side effect(s) should occur.

It will also be appreciated that the antibodies and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the antibodies and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an antibody may be administered concurrently withanother agent used to treat the same disorder), or they may achievedifferent effects (e.g., control of any adverse effects). As usedherein, additional therapeutic agents which are normally administered totreat or prevent a particular disease, or condition, are appropriate forthe disease, or condition, being treated. When multiple therapeutics areco-administered, dosages may be adjusted accordingly, as is recognizedin the pertinent art.

A fifth aspect of the invention involves a method for treating a diseaseor disorder associated with increased GREM1 expression, such as fibrosisor cancer. In certain embodiments, invention provides a method fortreating a patient suffering from cancer, or for treating at least onesymptom or complication associated with cancer, or halting theprogression of cancer, the method comprising administering to thepatient an effective amount of an antibody or an antigen-bindingfragment thereof that binds to human GREM1; or a pharmaceuticalcomposition comprising an effective amount of an antibody or anantigen-binding fragment thereof that binds to GREM1, such that thecancer-associated condition or disease is either prevented, or lessenedin severity and/or duration, or at least one symptom or complicationassociated with the condition or disease is prevented, or ameliorated,or that the frequency and/or duration of, or the severity of cancer isreduced.

In certain embodiments, invention provides a method for treating apatient suffering from fibrosis, or for treating at least one symptom orcomplication associated with fibrosis, or halting the progression offibrosis, or for treating a patient at risk for developing fibrosis, themethod comprising administering to the patient an effective amount of anantibody or an antigen-binding fragment thereof that binds to GREM1; ora pharmaceutical composition comprising an effective amount of anantibody or an antigen-binding fragment thereof that binds to GREM1,such that the fibrosis-associated condition or disease is eitherprevented, or lessened in severity and/or duration, or at least onesymptom or complication associated with the condition or disease isprevented, or ameliorated, or that the frequency and/or duration of, orthe severity of fibrosis is reduced. In one embodiment, the antibody isadministered therapeutically (administered after fibrosis has beenestablished and given throughout the course of the condition) to apatient suffering from fibrosis-associated condition or disease, orsuffering from at least one symptom or complication associated with thecondition or disease. In one embodiment, the antibody is administeredprophylactically (administered prior to development of the condition) toa patient at risk for developing fibrosis-associated condition ordisease, or at risk for developing at least one symptom or complicationassociated with fibrosis. For example, such “patients at risk fordeveloping fibrosis” include the elderly, or patients with a familyhistory, or smokers, or patients who have some underlying medicalcondition that may pre-dispose them to acquiring fibrosis such asdiabetes, or patients exposed to asbestos, wood, metal dust orchemicals, viral infections, certain medications, or cigarette smoke orpatients with chronic liver injuries like viral hepatitis, parasiticinfection, metabolic or autoimmune diseases, congenital abnormalitiesand drug and alcohol abuse. Other patients at risk for developingfibrosis include patients with chronic kidney disease, acute kidneyinjury, chronic hypertension, heart failure, kidney transplant,scleroderma, exposure to radiocontrast agent, chronic allergy, chronicasthma or lung transplant.

In another embodiment, the at least one symptom or complicationassociated with the fibrosis-associated condition or disease is selectedfrom the group consisting of shortness of breath, persistent dry hackingcough, pain, weight loss, nausea, loss of appetite, fluid accumulationin abdomen, swelling in legs, fatigue, pulmonary hypertension,hyperglycemia, renal injury, urinary tract infection, liver damage, lossof liver function, loss of renal function, hypertension, decrease inquality of life, reduced life expectancy and relapse of a condition ordisease associated with fibrosis. In some embodiments, the disease orcondition associated with fibrosis may be present in liver, kidney,lungs, skin, gut or muscle. In another embodiment, thefibrosis-associated condition or disease is selected from the groupcomprising pulmonary fibrosis, pulmonary hypertension, idiopathicpulmonary fibrosis, renal fibrosis, liver fibrosis, ischemic renalinjury, tubulointerstitial fibrosis, diabetic nephropathy,nephrosclerosis, and nephrotoxicity.

Embodiments of the invention relate to methods of protecting againstprogressive tissue damage, or inhibiting or reducing tissue degenerationin a patient suffering from fibrosis, the methods comprisingadministering to the patient an effective amount of an antibody or anantigen-binding fragment thereof that binds to GREM1; or apharmaceutical composition comprising an effective amount of an antibodyor an antigen-binding fragment thereof that binds to GREM1, such thatthe tissue in the patient is protected from progressive damage or tissuedegeneration is inhibited or reduced in a patient suffering fromfibrosis. In some embodiments of the invention, the tissue affected byfibrotic damage is lungs, wherein the fibrotic disease may be one ofpulmonary fibrosis, pulmonary hypertension or idiopathic pulmonaryfibrosis. In one embodiment, the tissue affected by fibrotic damage maybe liver. In some embodiments, the tissue affected by fibrotic damagemay be kidney, wherein the fibrotic disease may comprise one of renalfibrosis, ischemic renal injury, tubulointerstitial fibrosis, diabeticnephropathy, nephrosclerosis, or nephrotoxicity.

In some embodiments, the invention includes methods of treating canceror inhibiting tumor growth, tumor cell proliferation or tumormetastasis, the methods comprising administering an isolated antibody orantigen-binding fragment thereof of the present invention that binds toGREM1. In certain embodiments, the invention includes methods forinhibiting angiogenesis, the methods comprising administering anisolated antibody or antigen-binding fragment thereof of the presentinvention that binds to GREM1.

In one embodiment, the pharmaceutical composition comprising theantibodies of the invention is administered to the patient incombination with a second therapeutic agent.

In another embodiment, the second therapeutic agent is selected from thegroup consisting of an anti-fibrotic agent such as pirfenidone, ananti-inflammatory drug, a NSAID, a corticosteroid such as prednisone, anutritional supplement, a vascular endothelial growth factor (VEGF)antagonist [e.g., a “VEGF-Trap” such as aflibercept or otherVEGF-inhibiting fusion protein as set forth in U.S. Pat. No. 7,087,411,or an anti-VEGF antibody or antigen binding fragment thereof (e.g.,bevacizumab, or ranibizumab)], an antibody to a cytokine such as IL-1,IL-6, IL-13, IL-4, IL-17, IL-25, IL-33 or TGF-β, and any otherpalliative therapy useful for ameliorating at least one symptomassociated with a fibrosis-associated condition or cancer. In someembodiments, the second therapeutic agent may be administered to manageor treat at least one complication associated with fibrosis or cancer.

In embodiments of the invention, the antibody or antigen-bindingfragment thereof or the pharmaceutical composition comprising theantibody is administered subcutaneously, intravenously, intradermally,orally or intramuscularly.

In some embodiments, the antibody or antigen-binding fragment thereof isadministered at doses of about 0.1 mg/kg of body weight to about 100mg/kg of body weight, more specifically about 20 mg/kg of body weight toabout 50 mg/kg of body weight.

In related embodiments, the invention includes the use of an isolatedanti-GREM1 antibody or antigen binding portion of an antibody of theinvention in the manufacture of a medicament for the treatment of adisease or disorder related to or caused by GREM1 activity. In oneembodiment, the invention includes an isolated anti-GREM1 antibody orantigen-binding fragment thereof for use in promoting BMP signaling orcell differentiation. In one embodiment, the invention includes anisolated anti-GREM1 antibody or antigen-binding fragment thereof for usein inhibiting heparin-mediated angiogenesis. In one embodiment, theinvention includes the use of an anti-GREM1 antibody of the invention inthe manufacture of a medicament for treating a patient suffering from orat risk of developing fibrosis. In one embodiment, the inventionincludes the use of an anti-GREM1 antibody of the invention in themanufacture of a medicament for treating a patient suffering fromcancer.

A sixth aspect of the invention provides for methods of predictingprognosis of fibrosis in a patient suffering from a condition or diseaseselected from the group comprising of pulmonary fibrosis, idiopathicpulmonary fibrosis, pulmonary hypertension, renal fibrosis, hepaticfibrosis and diabetic nephropathy, the method comprising reacting aGREM1 protein from the patient with an antibody or antigen-bindingfragment of the invention, wherein binding with human GREM1 indicatespoor prognosis.

In one embodiment, the invention features a method of predicting poorsurvival in a patient suffering from fibrosis, the method comprisingreacting a GREM1 protein from the patient with an isolated antibody ofthe invention as described herein, wherein binding with GREM1 indicatespoor survival.

In one embodiment, the tissue or cell sample containing a GREM1 proteinfrom a patient is obtained from the patient's blood, serum, plasma, orbiopsy of a tissue, such as liver, lung or kidney.

In a related embodiment, the invention features a method of diagnosingfibrosis in a tissue or monitoring fibrotic activity in a subjectsuspected of suffering from fibrosis, the method comprisingadministering an antibody or antigen-binding fragment of the inventionlinked to a detectable label such as a radionuclide or a MRI-detectablelabel and imaging the subject upon such administration, wherein GREM1binding and detection in the image indicates fibrosis.

In one embodiment, the fibrosis is idiopathic pulmonary fibrosis. Insome embodiments, the fibrosis is selected from the group comprisingpulmonary hypertension, diabetic nephropathy, renal fibrosis, liverfibrosis, and tubulointerstitial fibrosis. In some embodiments, thefibrotic activity is detected in lungs or kidney or liver.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 describes the structural features of GREM1 protein (FromWordinger, R. J., et al 2008, Exp. Eye Res. 87: 78-79). The predictedpositions of structural features are shown. Signal Seq., signal sequence(positions 1-24); DAN, cysteine-rich motif (positions 69-184);

, glycosylation site (position 42); *, phosphorylation sites (positions6, 29, 44, 47, 55, 66, 76, 77, 88, 102 and 151);

, PKC specific eukaryotic protein phosphorylation site (position 165);NLS, nuclear localization signal sequences (positions 145, 166, 163,164).

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

Definitions

The term “bone morphogenetic protein” or “BMP” refers to the group ofgrowth factors which function as pivotal morphogenetic signals,orchestrating tissue architecture throughout the body. They wereoriginally discovered by their ability to induce the formation of boneand cartilage. However, BMPs have a variety of different functionsduring embryonic development. They are also involved in body patterningand morphogenesis cascades. BMPS have been found to be essential inorgan homeostasis. Further, BMPs play important roles in thepathophysiology of several diseases including osteoporosis, arthritis,pulmonary hypertension and kidney diseases. BMPs and their involvementin disease processes have been reviewed by Weiskirchen, R., et al inFront. Biosci. 2009, 14: 4992-5012. Twenty BMPs have been discovered sofar, of which BMP2 to BMP7 belong to the transforming growth factor betasuperfamily.

The term “GREM1” refers to human gremlin-1, a member of the cysteineknot superfamily. The amino acid sequence of human GREM1 is provided inGenBank as accession number NP_037504 and is also referred to herein asSEQ ID NO: 594. GREM1 is encoded by the nucleic acid provided herein asSEQ ID NO: 593, and is also found in GenBank as accession numberNM_013372. GREM1 is a highly conserved 184 aa protein which has beenmapped to chromosome 15q13-q15. The protein contains a signal peptide(aa 1-24) and a predicted glycosylation site (at aa 42). In addition,the protein contains a cysteine-rich region and a cysteine knot motif(aa 94-184) whose structure is shared by members of the transforminggrowth factor-beta (TGF-β) superfamily. GREM1 exists in both secretedand cell-associated (e.g. membrane associated) forms. GREM1 is alsoknown as gremlin1, cysteine knot superfamily 1-BMP antagonist 1(CKTSF1B1), DAN domain family member 2 (DAND2), Down-regulated inMos-transformed cells protein (DRM), gremlin, GREMLIN, Gremlin-1precursor, Increased in high glucose protein 2 (IHG-2), MGC126660,Proliferation-inducing gene 2 protein (PIG2), or Gremlin 1-like protein.GREM1 is an antagonist of bone morphogenetic proteins (BMPs). It bindsto BMPs and inhibits their binding to their receptors. The interplaybetween GREM1 and BMPs fine-tunes the level of available BMPs andaffects developmental and disease processes. GREM1 can bind to andinhibit BMP-2, BMP-4 and BMP-7. GREM1 has been found to be up regulatedin fibrotic diseases, especially of the kidney, lung and liver.

The term “fibrosis”, as used herein refers to the formation of excessfibrous connective tissue in an organ or tissue in a reparative orreactive process. This is as opposed to formation of fibrous tissue as anormal constituent of an organ or tissue. Scarring is confluent fibrosisthat obliterates the architecture of the underlying organ or tissue.Fibrosis can affect many organs in the body. The following table showssome examples of fibrosis along with the affected organ:

Type of fibrosis Organ affected Pulmonary fibrosis Lungs Cystic fibrosisLungs Idiopathic pulmonary fibrosis Lungs Cirrhosis (associated withviral Liver infection or other cause) Non-alcoholic steatohepatitisLiver Endomyocardial fibrosis Heart Mediastinal fibrosis Soft tissue ofthe mediastinum Myelofibrosis Bone marrow Retroperitoneal fibrosis Softtissue of the retroperitoneum Progressive massive fibrosis Lungs (acomplication of coal workers' pneumoconiosis) Bronchiolitis obliteransLungs Airway Remodeling associated Lungs with chronic asthma Kidney orLung transplant fibrosis Kidney, Lungs Focal & Segmental KidneyGlomerulosclerosis Nephrogenic systemic fibrosis Skin Crohn's diseaseIntestine Keloid Skin Old myocardial infarction Heart Muscular dystrophyMuscle Scleroderma, systemic sclerosis Skin, lungs Arthrofibrosis Knee,shoulder, other joints Corneal fibrosis Eyes Retinal fibrosis associatedwith Eyes macular degeneration

The term “fibrosis” also comprises complex disorders such as pulmonaryfibrosis, for example, idiopathic pulmonary fibrosis, pulmonaryhypertension, diabetic nephropathy, ischemic renal injury, renalfibrosis, hepatic fibrosis, tubulointerstitial fibrosis, nephrosclerosisand nephrotoxicity.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds(i.e., “full antibody molecules”), as well as multimers thereof (e.g.IgM) or antigen-binding fragments thereof. Each heavy chain is comprisedof a heavy chain variable region (“HCVR” or “V_(H)”) and a heavy chainconstant region (comprised of domains C_(H)1, C_(H)2 and C_(H)3). Eachlight chain is comprised of a light chain variable region (“LCVR or“V_(L)”) and a light chain constant region (C_(L)). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, theFRs of the antibody (or antigen binding fragment thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antibodies have been described in the scientificliterature in which one or two CDRs can be dispensed with for binding.Padlan et al. (FASEB J. 1995, 9:133-139) analyzed the contact regionsbetween antibodies and their antigens, based on published crystalstructures, and concluded that only about one fifth to one third of CDRresidues actually contact the antigen. Padlan also found many antibodiesin which one or two CDRs had no amino acids in contact with an antigen(see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues not contacting antigen can be identified based on previousstudies (for example residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The fully human anti-GREM1 monoclonal antibodies disclosed herein maycomprise one or more amino acid substitutions, insertions and/ordeletions in the framework and/or CDR regions of the heavy and lightchain variable domains as compared to the corresponding germlinesequences. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. The presentinvention includes antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes fully human anti-GREM1 monoclonalantibodies comprising variants of any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein having one or more conservativesubstitutions. For example, the present invention includes anti-GREM1antibodies having HCVR, LCVR, and/or CDR amino acid sequences with,e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservativeamino acid substitutions relative to any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human mAbs of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include mAbs in which CDR sequences derivedfrom the germline of another mammalian species (e.g., mouse), have beengrafted onto human FR sequences.

The term “specifically binds,” or “binds specifically to”, or the like,means that an antibody or antigen-binding fragment thereof forms acomplex with an antigen that is relatively stable under physiologicconditions. Specific binding can be characterized by an equilibriumdissociation constant of at least about 1×10⁻⁶ M or less (e.g., asmaller K_(D) denotes a tighter binding). Methods for determiningwhether two molecules specifically bind are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. As described herein, antibodies which bind specifically tohuman GREM1 have been identified by surface plasmon resonance, e.g.,BIACORE™. Moreover, multi-specific antibodies that bind to one domain inGREM1 and one or more additional antigens or a bi-specific that binds totwo different regions of GREM1 are nonetheless considered antibodiesthat “specifically bind”, as used herein.

The term “high affinity” antibody refers to those mAbs having a bindingaffinity to GREM1, expressed as K_(D), of at least 10⁻⁷ M; preferably10⁻⁸ M; more preferably 10⁻⁹M, even more preferably 10⁻¹⁰ M, even morepreferably 10⁻¹¹ M, as measured by surface plasmon resonance, e.g.,BIACORE™ or solution-affinity ELISA.

By the term “slow off rate”, “Koff” or “kd” is meant an antibody thatdissociates from GREM1, with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁻⁴ s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

The terms “antigen-binding portion” of an antibody, “antigen-bindingfragment” of an antibody, and the like, as used herein, include anynaturally occurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. The terms “antigen-binding fragment” of anantibody, or “antibody fragment”, as used herein, refers to one or morefragments of an antibody that retain the ability to bind to GREM1.

In specific embodiments, antibody or antibody fragments of the inventionmay be conjugated to a therapeutic moiety (“immunoconjugate”), such asan antibiotic, a second anti-GREM1 antibody, or an antibody to acytokine such as IL-1, IL-6, or TGF-β, or any other therapeutic moietyuseful for treating a disease or condition including pulmonary fibrosis,renal fibrosis, liver fibrosis, ischemic renal injury,tubulointerstitial fibrosis, diabetic nephropathy, nephrosclerosis, ornephrotoxicity.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies (Abs) havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds human GREM1, or a fragment thereof, is substantiallyfree of Abs that specifically bind antigens other than GREM1.

A “blocking antibody” or a “neutralizing antibody”, as used herein (oran “antibody that neutralizes GREM1 activity”), is intended to refer toan antibody whose binding to GREM1 results in inhibition of at least onebiological activity of GREM1. For example, an antibody of the inventionmay aid in inhibiting or preventing the spread of fibrosis.Alternatively, an antibody of the invention may demonstrate the abilityto treat fibrosis or at least one symptom caused by fibrosis, includingdry cough or breathlessness. This inhibition of the biological activityof GREM1 can be assessed by measuring one or more indicators of GREM1biological activity by one or more of several standard in vitro assays(such as a neutralization assay, as described herein) or in vivo assaysknown in the art (for example, animal models to look at protection fromGREM1 activity following administration of one or more of the antibodiesdescribed herein).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timebiomolecular interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIACORE™system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. The term“epitope” also refers to a site on an antigen to which B and/or T cellsrespond. It also refers to a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below. A nucleic acid molecule havingsubstantial identity to a reference nucleic acid molecule may, incertain instances, encode a polypeptide having the same or substantiallysimilar amino acid sequence as the polypeptide encoded by the referencenucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positions,which are not identical, differ by conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment are wellknown to those of skill in the art. See, e.g., Pearson (1994) MethodsMol. Biol. 24: 307-331, which is herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997)Nucleic Acids Res. 25: 3389-3402, each of which is herein incorporatedby reference.

In specific embodiments, the antibody or antibody fragment for use inthe method of the invention may be mono-specific, bi-specific, ormulti-specific. Multi-specific antibodies may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for epitopes of more than one target polypeptide. Anexemplary bi-specific antibody format that can be used in the context ofthe present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bi-specific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise an Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 mAbs;N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the caseof IgG2 mAbs; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 mAbs. Variations on the bi-specific antibody format describedabove are contemplated within the scope of the present invention.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

General Description

As an antagonist of bone morphogenetic proteins (BMPs), GREM1 gene playsa role in regulating organogenesis, body patterning, and tissuedifferentiation. GREM1 has been found to play an important role in lungdevelopment. However, expression of GREM1 in a healthy adult lung islow. Upregulated levels of GREM1 have been correlated with pulmonaryhypertension and pulmonary fibrosis (Costello, et al., 2010, Am. J.Respir. Cell. Mol. Biol. 42: 517-523). Pulmonary fibrosis, especially ofthe idiopathic type is a progressive, scar-forming and disabling diseaseof the lung parenchyma with a poor prognosis and no efficacious therapy.Elevated GREM1 expression correlates negatively with lung function testsin idiopathic pulmonary fibrosis, suggesting that GREM1 may be animportant marker of advanced stage fibrosis (Costello, et al., 2010, Am.J. Respir. Cell. Mol. Biol. 42: 517-523).

GREM1 expression is also essential in kidney organogenesis. However,GREM1 expression in a healthy adult kidney is almost undetectable.Elevated GREM1 levels are found in patients with hyperglycemia anddiabetic nephropathy (Lappin, et al., 2002, Nephrol. Dial. Transplant.17: 65-67). GREM1 is found to be upregulated in areas oftubulointerstitial fibrosis in patients with diabetic nephropathy.Diabetic nephropathy is a complex disorder characterized by sclerosisand development of tubulointerstitial fibrosis. It is the leading causeof end-stage renal diseases and 20-40% of patients with diabetesultimately develop diabetic nephropathy. Specific therapies to reverseor inhibit the progression of diabetic nephropathy to advanced stagesare not available and current treatment strategies are limited tomanagement of blood glucose levels and control of hypertension (Zhang etal., 2009, BBRC 383: 1-3).

GREM1 has also been found to be upregulated in liver fibrosis (Boers etal., 2006, J. Biol. Chem. 281: 16289-16295). Hepatic fibrosis is acommon response to most chronic liver injuries like viral hepatitis,parasitic infection, metabolic or autoimmune diseases, congenitalabnormalities and drug and alcohol abuse. Fibrosis may also contributeto progressive cirrhosis of liver. Detection of liver disease is oftendelayed and effective medical treatment is not readily available.

The antibodies described herein demonstrate specific binding to humanGREM1 and in some embodiments, may be useful for treating patientssuffering from fibrosis. The use of such antibodies may be an effectivemeans of treating patients suffering from fibrosis, or may be used tolessen the severity of the dry cough or difficulty in breathingassociated with fibrosis. They may be used alone or as adjunct therapywith other therapeutic moieties or modalities known in the art fortreating fibrosis, such as, but not limited to, a non-steroidalanti-inflammatory drug (NSIAD), a corticosteroid such as prednisone, orany other palliative therapy. They may be used in conjunction with asecond or third different antibody specific for GREM1, or against acytokine such as IL-1, IL-6 or TGF-β.

In some embodiments, the antibodies described herein may be useful intreating or managing a disease or condition of fibrosis including(idiopathic) pulmonary fibrosis, renal fibrosis, liver fibrosis,ischemic renal injury, tubulointerstitial fibrosis, diabeticnephropathy, nephrosclerosis, or nephrotoxicity.

In certain embodiments, the antibodies described herein may be usefulfor treating or managing cancer such as sarcoma, and carcinomas of thelung, uterine cervix, colon, breast, and pancreas.

In certain embodiments, the antibodies of the invention are obtainedfrom mice immunized with a primary immunogen, such as a native, fulllength human GREM1 (See GenBank accession number NP_037504 (SEQ ID NO:594)) or with a recombinant form of GREM1 (SEQ ID NO: 595) or GREM1fragments, followed by immunization with a secondary immunogen, or withan immunogenically active fragment of GREM1.

The immunogen may be an immunogenic fragment of human GREM1 or DNAencoding the fragment thereof. The immunogen may GREM1 coupled to ahistidine tag and/or to a fragment of Fc region of an antibody.

The amino acid sequence of full length human GREM1 (also known by Genbank accession number NP-037504) is shown as SEQ ID NO: 594. The fulllength amino acid sequence of recombinant GREM1 (aa 25-184 GREM1 coupledto Fc region and a histidine tag) is shown as SEQ ID NO: 595.

The full-length DNA sequence of GREM1 is shown as SEQ ID NO: 593.

In certain embodiments, antibodies that bind specifically to human GREM1may be prepared using fragments of the above-noted regions, or peptidesthat extend beyond the designated regions by about 5 to about 20 aminoacid residues from either, or both, the N or C terminal ends of theregions described herein. In certain embodiments, any combination of theabove-noted regions or fragments thereof may be used in the preparationof human GREM1 specific antibodies. In certain embodiments, any one ormore of the above-noted regions of human GREM1, or fragments thereof maybe used for preparing monospecific, bispecific, or multispecificantibodies.

Antigen-Binding Fragments of Antibodies

Unless specifically indicated otherwise, the term “antibody,” as usedherein, shall be understood to encompass antibody molecules comprisingtwo immunoglobulin heavy chains and two immunoglobulin light chains(i.e., “full antibody molecules”) as well as antigen-binding fragmentsthereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. The terms “antigen-binding fragment”of an antibody, or “antibody fragment”, as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to human GREM1. An antibody fragment may include a Fab fragment, aF(ab′)₂ fragment, a Fv fragment, a dAb fragment, a fragment containing aCDR, or an isolated CDR. Antigen-binding fragments of an antibody may bederived, e.g., from full antibody molecules using any suitable standardtechniques such as proteolytic digestion or recombinant geneticengineering techniques involving the manipulation and expression of DNAencoding antibody variable and (optionally) constant domains. Such DNAis known and/or is readily available from, e.g., commercial sources, DNAlibraries (including, e.g., phage-antibody libraries), or can besynthesized. The DNA may be sequenced and manipulated chemically or byusing molecular biology techniques, for example, to arrange one or morevariable and/or constant domains into a suitable configuration, or tointroduce codons, create cysteine residues, modify, add or delete aminoacids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR,which is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemono-specific or multi-specific (e.g., bi-specific). A multi-specificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multi-specific antibody format, including theexemplary bi-specific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind tohuman GREM1.

Using VELOCIMMUNE™ technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other knownmethod for generating monoclonal antibodies, high affinity chimericantibodies to human GREM1 are initially isolated having a human variableregion and a mouse constant region. The VELOCIMMUNE® technology involvesgeneration of a transgenic mouse having a genome comprising human heavyand light chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibody are isolated and operably linkedto DNA encoding the human heavy and light chain constant regions. TheDNA is then expressed in a cell capable of expressing the fully humanantibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. As in the experimentalsection below, the antibodies are characterized and selected fordesirable characteristics, including affinity, selectivity, epitope,etc. The mouse constant regions are replaced with a desired humanconstant region to generate the fully human antibody of the invention,for example wild-type or modified IgG1 or IgG4. While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

In general, the antibodies of the instant invention possess very highaffinities, typically possessing K_(D) of from about 10⁻¹² through about10⁻⁷ M, when measured by binding to antigen either immobilized on solidphase or in solution phase. The mouse constant regions are replaced withdesired human constant regions to generate the fully human antibodies ofthe invention. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Bioequivalents

The anti-human GREM1 antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies, but that retain the ability to bindhuman GREM1. Such variant antibodies and antibody fragments comprise oneor more additions, deletions, or substitutions of amino acids whencompared to parent sequence, but exhibit biological activity that isessentially equivalent to that of the described antibodies. Likewise,the antibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an antibody or antibody fragment that is essentiallybioequivalent to an antibody or antibody fragment of the invention.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single dose or multipledoses. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of the antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include antibody variants comprising aminoacid changes, which modify the glycosylation characteristics of theantibodies, e.g., mutations that eliminate or remove glycosylation.

Anti-GREM1 Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-GREM1antibodies are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-GREM1 antibodies comprising a mutationin the C_(H)2 or a C_(H)3 region of the Fc domain, wherein themutation(s) increases the affinity of the Fc domain to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0). Such mutations may result in an increase in serum half-lifeof the antibody when administered to an animal. Non-limiting examples ofsuch Fc modifications include, e.g., a modification at position 250(e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T),254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification atposition 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W,H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P). Inyet another embodiment, the modification comprises a 265A (e.g., D265A)and/or a 297A (e.g., N297A) modification.

For example, the present invention includes anti-GREM1 antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I andQ311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g.,D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A);and 433K and 434F (e.g., H433K and N434F). All possible combinations ofthe foregoing Fc domain mutations, and other mutations within theantibody variable domains disclosed herein, are contemplated within thescope of the present invention.

The present invention also includes anti-GREM1 antibodies comprising achimeric heavy chain constant (C_(H)) region, wherein the chimeric C_(H)region comprises segments derived from the C_(H) regions of more thanone immunoglobulin isotype. For example, the antibodies of the inventionmay comprise a chimeric C_(H) region comprising part or all of a C_(H)2domain derived from a human IgG1, human IgG2 or human IgG4 molecule,combined with part or all of a C_(H)3 domain derived from a human IgG1,human IgG2 or human IgG4 molecule. According to certain embodiments, theantibodies of the invention comprise a chimeric C_(H) region having achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” amino acid sequence (amino acid residues from positions216 to 227 according to EU numbering) derived from a human IgG1, a humanIgG2 or a human IgG4 hinge region, combined with a “lower hinge”sequence (amino acid residues from positions 228 to 236 according to EUnumbering) derived from a human IgG1, a human IgG2 or a human IgG4 hingeregion. According to certain embodiments, the chimeric hinge regioncomprises amino acid residues derived from a human IgG1 or a human IgG4upper hinge and amino acid residues derived from a human IgG2 lowerhinge. An antibody comprising a chimeric C_(H) region as describedherein may, in certain embodiments, exhibit modified Fc effectorfunctions without adversely affecting the therapeutic or pharmacokineticproperties of the antibody. (See, e.g., U.S. Provisional Appl. No.61/759,578, filed Feb. 1, 2013, the disclosure of which is herebyincorporated by reference in its entirety).

Biological Characteristics of the Antibodies

In general, the antibodies of the present invention may function bybinding to human GREM1. In some embodiments, the antibodies of thepresent invention may bind to the catalytic domain of human GREM1, or toa fragment thereof. In some embodiments, the antibodies of the inventionmay bind to the secreted form of human GREM1 or to themembrane-associated form of human GREM1. In some embodiments, theantibodies of the present invention may bind to more than one domain(cross-reactive antibodies).

In certain embodiments of the invention, the antibodies may bind to anepitope located in the region between amino acid residues 25-184 of SEQID NO: 594 or SEQ ID NO: 595.

In certain embodiments, the antibodies of the present invention mayfunction by blocking or inhibiting BMP signaling by binding to any otherregion or fragment of the full length native protein, the amino acidsequence of which is shown in SEQ ID NO: 594, which is encoded by thenucleic acid sequence shown in SEQ ID NO: 593. In one embodiment, theantibodies of the present invention may function by reversing theinhibition of BMP2, BMP4 or BMP7 by binding to full-length GREM1 or afragment thereof. In some embodiments, the antibodies of the presentinvention may function by promoting BMP signaling or may block thebinding between GREM1 and BMPs including BMP2, BMP4 or BMP7.

In certain embodiments, the antibodies of the present invention mayfunction by blocking GREM1 binding to heparin and/or by inhibitingheparin-mediated VEGFR-2 activation.

In certain embodiments, the antibodies of the present invention may bebi-specific antibodies. The bi-specific antibodies of the invention maybind one epitope in one domain and may also bind one epitope in a seconddomain of human GREM1. In certain embodiments, the bi-specificantibodies of the invention may bind two different epitopes in the samedomain.

In one embodiment, the invention provides a fully human monoclonalantibody or antigen-binding fragment thereof that binds to human GREM1,wherein the antibody or fragment thereof exhibits one or more of thefollowing characteristics: (i) comprises a HCVR having an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50,66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290,306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482, 498, 514,530, 546, 562, and 578, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; (ii) comprises a LCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106,122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330,346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554,570, and 586, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;(iii) comprises a HCDR3 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120,136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344,360, 376, 392, 408, 424, 440, 456, 472, 488, 504, 520, 536, 552, 568,and 584, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; and aLCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160,176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384,400, 416, 432, 448, 464, 480, 496, 512, 528, 544, 560, 576, and 592, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; (iv) comprises aHCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164,180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388,404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, and 580, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a HCDR2 domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214,230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438,454, 470, 486, 502, 518, 534, 550, 566, and 582, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; a LCDR1 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 12, 28, 44,60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284,300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476, 492, 508,524, 540, 556, 572, and 588, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; and a LCDR2 domain having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126,142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350,366, 382, 398, 414, 430, 446, 462, 478, 494, 510, 526, 542, 558, 574,and 590, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; (v)binds to GREM1 with a K_(D) equal to or less than 10⁻⁷; (vi) blocksGREM1 binding to one of BMP2, BMP4 or BMP7; (vii) blocks GREM1inhibition of BMP signaling and promotes cell differentiation; and(viii) blocks GREM1 binding to heparin.

Certain anti-GREM1 antibodies of the present invention are able to bindto and neutralize the activity of GREM1, as determined by in vitro or invivo assays. The ability of the antibodies of the invention to bind toand neutralize the activity of GREM1 may be measured using any standardmethod known to those skilled in the art, including binding assays, oractivity assays, as described herein.

Non-limiting, exemplary in vitro assays for measuring binding activityare illustrated in Example 4, herein. In Example 4, the bindingaffinities and kinetic constants of human anti-GREM1 antibodies weredetermined by surface plasmon resonance and the measurements wereconducted on a T200 Biacore instrument. In Example 5, blocking assayswere used to determine the ability of the anti-GREM1 antibodies to blockthe BMP4 binding ability of GREM1 in vitro. Examples 6 and 7 describethe activity of the anti-GREM1 antibodies in promoting BMP4 signalingand cell differentiation. In Example 6, the anti-GREM1 antibodiesblocked the GREM1 inhibition of BMP4 signaling. In Example 7, theanti-GREM1 antibodies promoted BMP4 signaling and cell differentiationof osteoblast progenitor cells. Example 9 describes inhibition of theGREM1-heparin binding interaction using GREM1-specific antibodies.

The present invention also includes anti-GREM1 antibodies and antigenbinding fragments thereof which bind to at least one biologically activefragment of any of the following proteins, or peptides: SEQ ID NO: 594(full length native human GREM1), or SEQ ID NO: 595 (recombinant form ofhuman GREM1). Any of the GREM1 peptides described herein, or fragmentsthereof, may be used to generate anti-GREM1 antibodies.

The peptides may be modified to include addition or substitution ofcertain residues for tagging or for purposes of conjugation to carriermolecules, such as, KLH. For example, a cysteine may be added at eitherthe N terminal or C terminal end of a peptide, or a linker sequence maybe added to prepare the peptide for conjugation to, for example, KLH forimmunization.

The antibodies specific for GREM1 may contain no additional labels ormoieties, or they may contain an N-terminal or C-terminal label ormoiety. In one embodiment, the label or moiety is biotin. In a bindingassay, the location of a label (if any) may determine the orientation ofthe peptide relative to the surface upon which the peptide is bound. Forexample, if a surface is coated with avidin, a peptide containing anN-terminal biotin will be oriented such that the C-terminal portion ofthe peptide will be distal to the surface. In one embodiment, the labelmay be a radionuclide, a fluorescent dye or a MRI-detectable label. Incertain embodiments, such labeled antibodies may be used in diagnosticassays including imaging assays.

Epitope Mapping and Related Technologies

The present invention includes anti-GREM1 antibodies which interact withone or more amino acids found within one or more regions of GREM1. Theepitope to which the antibodies bind may consist of a single contiguoussequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more) amino acids located within any of theaforementioned regions of the GREM1 molecule (e.g. a linear epitope in adomain). Alternatively, the epitope may consist of a plurality ofnon-contiguous amino acids (or amino acid sequences) located withineither or both of the aforementioned regions of the GREM1 molecule (e.g.a conformational epitope).

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,for example, routine cross-blocking assays, such as that described inAntibodies, Harlow and Lane (Cold Spring Harbor Press, Cold SpringHarbor, N.Y.). Other methods include alanine scanning mutationalanalysis, peptide blot analysis (Reineke (2004) Methods Mol Biol248:443-63), peptide cleavage analysis crystallographic studies and NMRanalysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Protein Science 9: 487-496). Another method that can be used toidentify the amino acids within a polypeptide with which an antibodyinteracts is hydrogen/deuterium exchange detected by mass spectrometry.In general terms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water and exchangeable protons within aminoacids that are protected by the antibody complex undergodeuterium-to-hydrogen back-exchange at a slower rate than exchangeableprotons within amino acids that are not part of the interface. As aresult, amino acids that form part of the protein/antibody interface mayretain deuterium and therefore exhibit relatively higher mass comparedto amino acids not included in the interface. After dissociation of theantibody, the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (seeUS 2004/0101920, herein specifically incorporated by reference in itsentirety). Each category may reflect a unique epitope either distinctlydifferent from or partially overlapping with epitope represented byanother category. This technology allows rapid filtering of geneticallyidentical antibodies, such that characterization can be focused ongenetically distinct antibodies. When applied to hybridoma screening,MAP may facilitate identification of rare hybridoma clones that producemAbs having the desired characteristics. MAP may be used to sort theantibodies of the invention into groups of antibodies binding differentepitopes.

In certain embodiments, the anti-GREM1 antibodies or antigen-bindingfragments thereof bind an epitope within any one or more of the regionsexemplified in GREM1, either in natural form, as exemplified in SEQ IDNO: 594, or recombinantly produced, as exemplified in SEQ ID NO: 595, orto a fragment thereof. In certain embodiments, the antibodies of theinvention, as shown in Table 1, interact with at least one amino acidsequence selected from the group consisting of amino acid residuesranging from about position 1 to about position 24 of SEQ ID NO: 594; oramino acid residues ranging from about position 25 to about position 184of SEQ ID NO: 594. These regions are further exemplified in SEQ ID NO:595.

The present invention includes anti-human GREM1 antibodies that bind tothe same epitope, or a portion of the epitope, as any of the specificexemplary antibodies described herein in Table 1, or an antibody havingthe CDR sequences of any of the exemplary antibodies described inTable 1. Likewise, the present invention also includes anti-human GREM1antibodies that compete for binding to GREM1 or a GREM1 fragment withany of the specific exemplary antibodies described herein in Table 1, oran antibody having the CDR sequences of any of the exemplary antibodiesdescribed in Table 1.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-GREM1 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-GREM1antibody of the invention, the reference antibody is allowed to bind toa GREM1 protein or peptide under saturating conditions. Next, theability of a test antibody to bind to the GREM1 molecule is assessed. Ifthe test antibody is able to bind to GREM1 following saturation bindingwith the reference anti-GREM1 antibody, it can be concluded that thetest antibody binds to a different epitope than the reference anti-GREM1antibody. On the other hand, if the test antibody is not able to bind tothe GREM1 protein following saturation binding with the referenceanti-GREM1 antibody, then the test antibody may bind to the same epitopeas the epitope bound by the reference anti-GREM1 antibody of theinvention.

To determine if an antibody competes for binding with a referenceanti-GREM1 antibody, the above-described binding methodology isperformed in two orientations: In a first orientation, the referenceantibody is allowed to bind to a GREM1 protein under saturatingconditions followed by assessment of binding of the test antibody to theGREM1 molecule. In a second orientation, the test antibody is allowed tobind to a GREM1 molecule under saturating conditions followed byassessment of binding of the reference antibody to the GREM1 molecule.If, in both orientations, only the first (saturating) antibody iscapable of binding to the GREM1 molecule, then it is concluded that thetest antibody and the reference antibody compete for binding to GREM1.As will be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antibody may notnecessarily bind to the identical epitope as the reference antibody, butmay sterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have thesame epitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other. Two antibodies have overlapping epitopes if some aminoacid mutations that reduce or eliminate binding of one antibody reduceor eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

Immunoconjugates

The invention encompasses a human anti-GREM1 monoclonal antibodyconjugated to a therapeutic moiety (“immunoconjugate”), such as an agentthat is capable of reducing the severity of fibrosis, or to ameliorateat least one symptom associated with fibrosis, including dry persistentcough and/or difficulty in breathing, or the severity thereof. As usedherein, the term “immunoconjugate” refers to an antibody which ischemically or biologically linked to a radioactive agent, a cytokine, aninterferon, a target or reporter moiety, an enzyme, a toxin, or atherapeutic agent. The antibody may be linked to the radioactive agent,cytokine, interferon, target or reporter moiety, enzyme, toxin, ortherapeutic agent at any location along the molecule so long as it isable to bind its target. An example of immunoconjugate is antibody drugconjugate. In some embodiments, the agent may be a second differentantibody to human GREM1, or to a cytokine such as IL-1, IL-6, or achemokine such as TGF-β. The type of therapeutic moiety that may beconjugated to the anti-GREM1 antibody and will take into account thecondition to be treated and the desired therapeutic effect to beachieved. For example, if the desired therapeutic effect is to treat thesequelae or symptoms associated with fibrosis, or any other conditionresulting from fibrosis, such as, but not limited to, inflammation orweight loss, it may be advantageous to conjugate an agent appropriate totreat the sequelae or symptoms of the condition, or to alleviate anyside effects of the antibodies of the invention. Examples of suitableagents for forming immunoconjugates are known in the art; see forexample, WO 05/103081. The preparation of immunoconjugates andimmunotoxins is generally well known in the art (see, e.g., U.S. Pat.No. 4,340,535). Immunoconjugates are described in detail, for example,in U.S. Pat. Nos. 7,250,492, 7,420,040 and 7,411,046, each of which isincorporated herein in their entirety.

Multi-Specific Antibodies

The antibodies of the present invention may be mono-specific,bi-specific, or multi-specific. Multi-specific antibodies may bespecific for different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The antibodies of the present inventioncan be linked to or co-expressed with another functional molecule, e.g.,another peptide or protein. For example, an antibody or fragment thereofcan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment to produce abi-specific or a multi-specific antibody with a second bindingspecificity. For example, the present invention includes bi-specificantibodies wherein one arm of an immunoglobulin is specific for theN-terminal region of GREM1, or a fragment thereof, and the other arm ofthe immunoglobulin is specific for the C-terminal region of GREM1, or asecond therapeutic target, or is conjugated to a therapeutic moiety. Anexemplary bi-specific antibody format that can be used in the context ofthe present invention involves the use of a first immunoglobulin (Ig)C_(H3) domain and a second Ig C_(H3) domain, wherein the first andsecond Ig C_(H3) domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bi-specific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H3) domain binds Protein A and the second Ig C_(H3) domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H3) may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H3) include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mab² bispecific formats (see, e.g., Klein et al. 2012,mAbs 4:6, 1-11, and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theanti-GREM1 antibodies or antigen-binding fragments thereof of thepresent invention. The administration of therapeutic compositions inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose of antibody may vary depending upon the age and the size of asubject to be administered, target disease, conditions, route ofadministration, and the like. When the antibody of the present inventionis used for treating fibrosis in an adult patient, or for treatingpulmonary hypertension, or for lessening the severity of the disease, itis advantageous to intravenously administer the antibody of the presentinvention normally at a single dose of about 0.1 to about 100 mg/kg bodyweight, more preferably about 5 to about 100, about 10 to about 90, orabout 20 to about 70 mg/kg body weight. Depending on the severity of thecondition, the frequency and the duration of the treatment can beadjusted. In certain embodiments, the antibody or antigen-bindingfragment thereof of the invention can be administered as an initial doseof at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about5 to about 300 mg, or about 10 to about 200 mg, to about 100 mg, or toabout 50 mg. In certain embodiments, the initial dose may be followed byadministration of a second or a plurality of subsequent doses of theantibody or antigen-binding fragment thereof in an amount that can beapproximately the same or less than that of the initial dose, whereinthe subsequent doses are separated by at least 1 day to 3 days; at leastone week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, transdermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. The pharmaceutical composition can be also deliveredin a vesicle, in particular a liposome (see, for example, Langer (1990)Science 249:1527-1533).

The use of nanoparticles to deliver the antibodies of the presentinvention is also contemplated herein. Antibody-conjugated nanoparticlesmay be used both for therapeutic and diagnostic applications.Antibody-conjugated nanoparticles and methods of preparation and use aredescribed in detail by Arruebo, M., et al. 2009 (“Antibody-conjugatednanoparticles for biomedical applications” in J. Nanomat. Volume 2009,Article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporatedherein by reference. Nanoparticles for drug delivery have also beendescribed in, for example, U.S. Pat. Nos. 8,277,812, 8,258,256,8,257,740, 8,246,995, 8,236,330, each incorporated herein in itsentirety.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used.In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (Sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier,Stuttgart, Germany), the EPIPEN (Dey, L. P.) and the HUMIRA™ Pen (AbbottLabs, Abbott Park, Ill.), to name only a few.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

In certain embodiments of the invention, the present antibodies areuseful for treating a disease or condition associated with fibrosis, orat least one symptom associated with the disease or condition, such aspersistent cough, breathlessness, weight loss or loss of appetite, orfor lessening the severity of the disease. In some embodiments, theantibodies may be useful for treating a condition or symptom of fibrosisat a later stage in the disease. The antibodies of the invention arealso contemplated for prophylactic use in patients at risk fordeveloping fibrosis. These patients include the elderly, or patientswith a family history, or patients immunocompromised due to illness ortreatment with immunosuppressive therapeutics, or patients who may havean underlying medical condition such as diabetes that predisposes themto fibrosis, or patients who may be predisposed to fibrosis due tolifestyle choices such as smoking or alcohol abuse. It is contemplatedthat the antibodies of the invention may be used alone, or inconjunction with a second agent, or third agent for treating fibrosis,or for alleviating at least one symptom or complication associated withfibrosis, such loss of kidney function or liver function associatedwith, or resulting from fibrosis. The second or third agents may bedelivered concurrently with the antibodies of the invention, or they maybe administered separately, either before or after the antibodies of theinvention.

Symptoms for fibrosis disorders include, but are not limited to, drycough, difficulty in breathing, loss of appetite, weight loss, fatigue,nausea, swelling and fluid accumulation, liver damage, liver failure,hypertension, and loss of renal function. Other signs or symptomsinclude, but are not limited to, malaise, poor sleep, or complicationssuch as pneumonia or urinary tract infection, hyperglycemia, andproteinuria. The antibodies of the present invention may be used torelieve or to prevent or to decrease the severity of one or more of thesymptoms or conditions listed above.

In certain embodiments, the present antibodies are useful for treating acondition or indication associated with cancer including, but notlimited to sarcoma, or carcinoma of lung, ovary, kidney, breast, colon,pancreas and uterine cervix.

In certain embodiments, one or more antibodies of the present inventionmay be used alone or in combination to block GREM1 binding to heparinand/or heparin-mediated angiogenesis.

In a further embodiment of the invention the present antibodies are usedfor the preparation of a pharmaceutical composition for treatingpatients suffering from fibrosis or cancer, or a symptom associated withfibrosis or cancer. In yet another embodiment of the invention thepresent antibodies are used for the preparation of a pharmaceuticalcomposition for reducing the tissue damage or for preventing progressivedegeneration or for protecting kidney function or liver function infibrosis. In one embodiment of the invention the present antibodies areused as adjunct therapy with any other agent useful for treatingfibrosis or cancer, including an analgesic, a NSAID, an anti-tumor drug,chemotherapy, radiotherapy, a glucocorticoid, a vascular endothelialgrowth factor (VEGF) antagonist [e.g., a “VEGF-Trap” such as afliberceptor other VEGF-inhibiting fusion protein as set forth in U.S. Pat. No.7,087,411, or an anti-VEGF antibody or antigen binding fragment thereof(e.g., bevacizumab, or ranibizumab)], a second antibody to GREM1, anantibody to GREM2 or to an inflammatory cytokine such as IL-1, IL-6, orTGF-β, or any other palliative therapy known to those skilled in theart.

Combination Therapies

Combination therapies may include an anti-GREM1 antibody of theinvention and any additional therapeutic agent that may beadvantageously combined with an antibody of the invention, or with abiologically active fragment of an antibody of the invention.

For example, a second or third therapeutic agent such as a non-steroidalanti-inflammatory agent (NSAID) or an analgesic may be employed to aidin alleviating the symptoms of fibrosis such as dry cough or difficultyin breathing. An example of a common analgesic is acetaminophen.Exemplary NSAIDs include aspirin, ibuprofen, and naproxen. Theadditional therapeutic agent may be an antibiotic to treat acomplication such as urinary tract infection. The antibodies of thepresent invention may be combined with an antihypertensive agent to slowdown the development of fibrosis. For example, for patients sufferingfrom diabetic nephropathy, the antibodies may be combined with treatmentsuch as angiotensin-converting enzyme inhibitors to reduce bloodpressure and protect kidney function.

The antibodies may be used in conjunction with other therapies, such ascorticosteroids, or nutritional supplements in fibrosis treatment.Anti-fibrotic drugs such as pirfenidone have been found both to producedurable symptomatic remissions and to delay or halt progression offibrosis. This is important as such damage is usually irreversible.Anti-inflammatories and analgesics improve pain but do not preventtissue damage or slow the disease progression. In some embodiments,second or third therapeutic agents may be used to minimize clinicalsymptoms such as nausea and swelling, as well as prevent fibrotic tissuedamage. An additional therapeutic agent may comprise cortisone therapy,e.g., a low dosage of prednisone or prednisolone may be used inconjunction with one or more antibodies of the present invention in along term treatment plan for fibrosis. The use of one or more antibodiesdirected to a cytokine such as IL-1, IL-6, or TGF-β in fibrosistreatments are also envisaged within the scope of the present invention.The antibodies of the present invention may be combined with additionaltherapeutic agents to minimize or prevent complications such as urinarytract infection, hyperglycemia or blood pressure.

The antibodies of the present invention may also be administered incombination with other treatment options for fibrosis including physicaltherapy, lifestyle changes (including exercise and weight control),pulmonary rehabilitation, oxygen therapy, and dietary changes.Transplant surgery of liver, lungs or kidney may be required in advancedforms of fibrosis.

The antibodies of the present invention may be combined synergisticallywith one or more anti-cancer drugs or therapy used to treat cancer.Examples of anti-cancer drugs and therapy that may be used include, butare not limited to, cytotoxins, chemotherapeutic agents, radiation andsurgery. In some embodiments, one or more antibodies of the presentinvention may be used in combination with an anti-inflammatory drug(e.g., corticosteroids, and non-steroidal anti-inflammatory drugs), anantibody to a tumor-specific antigen (e.g., CA9, CA125,melanoma-associated antigen (MAGE), carcinoembryonic antigen (CEA),vimentin, tumor-M2-PK, prostate-specific antigen (PSA), MART-1, andCA19-9), a vascular endothelial growth factor (VEGF) antagonist [e.g., a“VEGF-Trap” such as aflibercept or other VEGF-inhibiting fusion proteinas set forth in U.S. Pat. No. 7,087,411, or an anti-VEGF antibody orantigen binding fragment thereof (e.g., bevacizumab, or ranibizumab)], adietary supplement such as anti-oxidants or any palliative care to treatcancer.

The additional therapeutically active component(s) may be administeredprior to, concurrent with, or after the administration of the anti-GREM1antibody of the present invention. For purposes of the presentdisclosure, such administration regimens are considered theadministration of an anti-GREM1 antibody “in combination with” a secondtherapeutically active component.

Diagnostic Uses of the Antibodies

The anti-GREM1 antibodies of the present invention may also be used todetect and/or measure GREM1 in a sample, e.g., for diagnostic purposes.It is envisioned that any one or more of the antibodies of the inventionmay be used to detect severity of tissue damage in fibrosis. Exemplarydiagnostic assays for GREM1 may comprise, e.g., contacting a sample,obtained from a patient, with an anti-GREM1 antibody of the invention,wherein the anti-GREM1 antibody is labeled with a detectable label orreporter molecule or used as a capture ligand to selectively isolateGREM1 from patient samples. Alternatively, an unlabeled anti-GREM1antibody can be used in diagnostic applications in combination with asecondary antibody which is itself detectably labeled. The detectablelabel or reporter molecule can be a radioisotope, such as ³H, ¹⁴C, ³²P,³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such asfluorescein isothiocyanate, or rhodamine; or an enzyme such as alkalinephosphatase, β-galactosidase, horseradish peroxidase, or luciferase.Specific exemplary assays that can be used to detect or measure GREM1 ina sample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in GREM1 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient, which contains detectable quantities of either GREM1 protein,or fragments thereof, under normal or pathological conditions.Generally, levels of GREM1 in a particular sample obtained from ahealthy patient (e.g., a patient not afflicted with fibrosis) will bemeasured to initially establish a baseline, or standard, level of GREM1.This baseline level of GREM1 can then be compared against the levels ofGREM1 measured in samples obtained from individuals suspected of havingfibrosis related condition, or symptoms associated with such condition.

The antibodies specific for GREM1 may contain no additional labels ormoieties, or they may contain an N-terminal or C-terminal label ormoiety. In one embodiment, the label or moiety is biotin. In a bindingassay, the location of a label (if any) may determine the orientation ofthe peptide relative to the surface upon which the peptide is bound. Forexample, if a surface is coated with avidin, a peptide containing anN-terminal biotin will be oriented such that the C-terminal portion ofthe peptide will be distal to the surface. In some embodiments, thelabel may be detectable label such as a radionuclide, a fluorescent dyeor a MRI-detectable label. Detectable labels may be linked to theantibodies wherein such antibodies may be used in imaging assays.Methods using imaging assays may be useful for fibrosis diagnosis andprognosis, or monitoring fibrotic activity.

Aspects of the invention relate to use of the disclosed antibodies asmarkers for predicting prognosis of fibrosis in patients. GREM1 has beenfound to be upregulated in fibrotic tissues in, for example, lung orliver or kidneys. Elevated levels of GREM1 have been correlated todiabetic nephropathy or pulmonary hypertension and could be used forevaluation of patient's prognosis. Antibodies of the present inventionmay be used in diagnostic assays to evaluate prognosis of fibroticdisease in a patient and to predict survival.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1. Generation of Human Antibodies to Human GREM1

In certain embodiments, the immunogen may be a peptide from the Nterminal or C terminal end of human GREM1. In certain embodiments of theinvention, the immunogen is the mature protein of human GREM1 thatranges from about amino acid residues 25-184 of SEQ ID NO: 594. In oneembodiment, the antibodies of the invention were obtained from miceimmunized with full length recombinant human GREM1.

In certain embodiments, antibodies that bind specifically to human GREM1may be prepared using fragments of the above-noted regions, or peptidesthat extend beyond the designated regions by about 5 to about 20 aminoacid residues from either, or both, the N or C terminal ends of theregions described herein. In certain embodiments, any combination of theabove-noted regions or fragments thereof may be used in the preparationof GREM1 specific antibodies. In certain embodiments, any one or more ofthe above-noted domains of hGREM1, or fragments thereof may be used forpreparing monospecific, bispecific, or multispecific antibodies (seeExample 8 below for details).

The full length proteins, or fragments thereof, that were used asimmunogens, as noted above, were administered directly, with an adjuvantto stimulate the immune response, to a VELOCIMMUNE® mouse comprising DNAencoding human Immunoglobulin heavy and kappa light chain variableregions. The antibody immune response was monitored by a GREM1-specificimmunoassay. When a desired immune response was achieved splenocyteswere harvested and fused with mouse myeloma cells to preserve theirviability and form hybridoma cell lines. The hybridoma cell lines werescreened and selected to identify cell lines that produce GREM1-specificantibodies. Using this technique, and the various immunogens describedabove, several anti-GREM1, as well as cross-reactive, chimericantibodies (i.e., antibodies possessing human variable domains and mouseconstant domains) were obtained; exemplary antibodies generated in thismanner were designated as H1M2907N, H2M2780N, H2M2782N, H2M2783N,H4H2783N2, H2M2784N, H2M2785N, H2M2786N, H2M2889N, H2M2890N, H2M2891N,H2M2892N, H2M2895N, H2M2897N, H2M2898N, H2M2899N, H2M2901N, H2M2906N,H2M2926N, H3M2788N, and H3M2929N.

Anti-GREM1 antibodies were also isolated directly from antigen-positiveB cells without fusion to myeloma cells, as described in U.S.2007/0280945A1, herein specifically incorporated by reference in itsentirety. Using this method, several fully human anti-GREM1 antibodies(i.e., antibodies possessing human variable domains and human constantdomains) were obtained; exemplary antibodies generated in this mannerwere designated as follows: H4H6232P, H4H6233P, H4H6236P, H4H6238P,H4H6240P, H4H6243P, H4H6245P, H4H6246P, H4H6248P, H4H6250P, H4H6251P,H4H6252S, H4H6256P, H4H6260P, H4H6269P, and H4H6270P.

The biological properties of the exemplary antibodies generated inaccordance with the methods of this Example are described in detail inthe Examples set forth below.

Example 2. Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs of selected antibodies specific for human GREM1 and theircorresponding antibody identifiers. Antibodies are typically referred toherein according to the following nomenclature: Fc prefix (e.g. “H4H”,“H1M, “H2M”), followed by a numerical identifier (e.g. “2907” as shownin Table 1), followed by a “P” or “N” suffix. Thus, according to thisnomenclature, an antibody may be referred to as, e.g. “H1H2907”. TheH4H, H1M, and H2M prefixes on the antibody designations used hereinindicate the particular Fc region of the antibody. For example, an “H2M”antibody has a mouse IgG2 Fc, whereas an “H4H” antibody has a human IgG4Fc. As will be appreciated by a person of ordinary skill in the art, anH1M or H2M antibody can be converted to an H4H antibody, and vice versa,but in any event, the variable domains (including the CDRs), which areindicated by the numerical identifiers shown in Table 1, will remain thesame. Antibodies having the same numerical antibody designation, butdiffering by a letter suffix of N, B or P refer to antibodies havingheavy and light chains with identical CDR sequences but with sequencevariations in regions that fall outside of the CDR sequences (i.e., inthe framework regions). Thus, N, B and P variants of a particularantibody have identical CDR sequences within their heavy and light chainvariable regions but differ from one another within their frameworkregions.

TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVRLCDR1 LCDR2 LCDR3 2907N 2 4 6 8 10 12 14 16 2780N 18 20 22 24 26 28 3032 2782N 34 36 38 40 42 44 46 48 2783N 50 52 54 56 58 60 62 64 2783N2 6668 70 72 74 76 78 80 2784N 82 84 86 88 90 92 94 96 2785N 98 100 102 104106 108 110 112 2786N 114 116 118 120 122 124 126 128 2889N 130 132 134136 138 140 142 144 2890N 146 148 150 152 154 156 158 160 2891N 162 164166 168 170 172 174 176 2892N 178 180 182 184 186 188 190 192 2895N 194196 198 200 202 204 206 208 2897N 210 212 214 216 218 220 222 224 2898N226 228 230 232 234 236 238 240 2899N 242 244 246 248 250 252 254 2562901N 258 260 262 264 266 268 270 272 2906N 274 276 278 280 282 284 286288 2926N 290 292 294 296 298 300 302 304 2788N 306 308 310 312 314 316318 320 2929N 322 324 326 328 330 332 334 336 6232P 338 340 342 344 346348 350 352 6233P 354 356 358 360 362 364 366 368 6236P 370 372 374 376378 380 382 384 6238P 386 388 390 392 394 396 398 400 6240P 402 404 406408 410 412 414 416 6243P 418 420 422 424 426 428 430 432 6245P 434 436438 440 442 444 446 448 6246P 450 452 454 456 458 460 462 464 6248P 466468 470 472 474 476 478 480 6250P 482 484 486 488 490 492 494 496 6251P498 500 502 504 506 508 510 512 6252P 514 516 518 520 522 524 526 5286256P 530 532 534 536 538 540 542 544 6260P 546 548 550 552 554 556 558560 6269P 562 564 566 568 570 572 574 576 6270P 578 580 582 584 586 588590 592

Example 3. Variable Gene Utilization Analysis

To analyze the structure of antibodies produced, the nucleic acidsencoding antibody variable regions were cloned and sequenced. From thenucleic acid sequence and predicted amino acid sequence of theantibodies, gene usage was identified for each Heavy Chain VariableRegion (HCVR) and Light Chain Variable Region (LCVR). Table 2 sets forththe gene usage for selected antibodies in accordance with the invention.

TABLE 2 HCVR LCVR Antibody V_(H) D_(H) J_(H) V_(K) J_(K) H4H2780N 3-211-26 6 1-27 3 H2bM2782N 3-21 1-7 4 1-27 3 H4H2784N 3-33 3-10 4 3-15 2H2bM2785N 3-23 6-13 3 1-33 2 H2bM2786N 3-23 2-8 3 1-33 2 H2M2783N 3-236-13 3 1-33 2 H4H2783N2 3-23 6-13 3 1-33 2 H3M2788N 3-33 4-23 4 1-17 1H4H2897N 3-30 2-21 4 2-28 3 H2bM2891N 3-7 3-3 4 1-12 2 H2AM2898N 3-132-2 6 1-6 1 H2BM2906N 3-7 3-3 4 1-12 2 H4H2892N 3-33 1-1 2 1-9 1H1M2907N 3-33 1-1 3 3-11 2 H2BM2890N 3-33 1-14 4 3-11 4 H2AM2899N 1-86-6 4 1-33 3 H4H2895N 3-53 3-9 4 6-21 1 H4H2926N 3-9 6-13 4 1-33 4H3M2929N 3-33 3-10 4 3-15 1 H2AM2901N 3-21 2-12 4 1-27 3 H4H2889N 4-594-4 6 1-6 1 H4H6232P 1-24 3-9 6 1-39 3 H4H6233P 3-33 1-7 4 3-11 2H4H6236P 1-24 3-9 6 or 3 1-39 3 H4H6238P 3-11 1-1 4 3-15 2 H4H6240P 3-331-7 4 3D-15 3 H4H6243P 1-24 3-9 6 or 4 1-39 3 H4H6245P 3-7 2-15 4 1-33 2H4H6246P 3-33 4-17 4 1-17 3 H4H6248P 3-33 4-17 4 3-15 1 H4H6250P 3-334-17 4 1-17 3 H4H6251P 3-33 4-17 4 3-15 1 H4H6252P 3-33 4-17 4 1-17 3H4H6256P 3-33 4-17 5 or 4 3-15 1 H4H6260P 3-33 1-1 3 3-11 5 H4H6269P4-31 1-1 4 1-6 4 H4H6270P 3-33 3-10 4 3-15 2

Example 4. Antibody Binding to Human GREM1 as Determined by SurfacePlasmon Resonance

Binding associative and dissociative rate constants (k_(a) and k_(d),respectively) and calculated equilibrium dissociation constants anddissociative half-lives (K_(D) and t_(1/2), respectively) for antigenbinding to purified anti-Gremlin1 (GREM1) antibodies were determinedusing a real-time surface plasmon resonance biosensor (Biacore T200)assay at 25° C. and at 37° C.

Anti-GREM1 antibodies were captured on either a goat anti-mouse IgGpolyclonal antibody (GE Healthcare, #BR-1008-38) or a mouse anti-humanIgG monoclonal antibody (GE Healthcare, #BR-1008-39) surface createdthrough direct amine coupling to a Biacore CM5 sensor chip. Kineticexperiments were carried out using HBS-EP+heparin [10 mM HEPES, 150 mMNaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20, 10 μg/ml heparin sodiumsalt, pH 7.4] as both the running buffer and the sample buffer.Antigen-antibody association rates were measured by injecting variousconcentrations (ranging from 11 to 100 nM, 3-fold dilutions) of humanGREM1 (hGREM1-His; SEQ ID NO: 595) over the captured anti-GREM1 antibodysurface. Antigen-antibody association was monitored for 150 secondswhile dissociation in buffer was monitored for 420 seconds. Kineticanalysis was performed using Scrubber software version 2.0a or BiacoreT200 evaluation software v1.0 to determine k_(a) and k_(d) values. K_(D)and t_(1/2) were then calculated from the experimentally determinedk_(a) and k_(d) values as K_(D)=k_(d)/k_(a) and t_(1/2)=In(2)/k_(d).

As shown in Table 3, thirty-five anti-GREM1 antibodies when captured onthe Biacore sensor exhibited binding to hGREM1-His protein injected overthe surface at 25° C., with K_(D) values ranging from 625 pM to 270 nM.Two of the antibodies tested, H2aM2898N and H2bM2785N, did not bindhGREM1-His under these experimental conditions. A subset of the 37anti-GREM1 antibodies was tested again at 37° C. As shown in Table 4,twenty-four anti-GREM1 antibodies when captured on the Biacore sensorexhibited binding to hGREM1-His protein injected over the surface at 37°C., with K_(D) values ranging from 1.23 nM to 275 nM.

TABLE 3 Biacore affinities at 25° C. for hGREM1-His binding to capturedanti-GREM1 monoclonal antibodies k_(a) k_(d) K_(D) t_(1/2) mAb captured(1/Ms) (1/s) (M) (min) H4H2895N 1.70E+05 1.32E−04 7.78E−10 88 H4H2780N7.12E+04 8.80E−04 1.24E−08 13 H4H2783N2 3.04E+04 4.76E−04 1.57E−08 24H4H2784N 6.18E+04 1.05E−03 1.70E−08 11 H4H2897N 2.77E+03 4.10E−041.48E−07 28 H4H2889N 6.27E+04 1.18E−04 1.89E−09 98 H4H2892N 1.42E+051.73E−04 1.22E−09 67 H4H2926N 9.10E+04 5.02E−04 5.52E−09 23 H4H6232P7.68E+04 9.60E−05 1.25E−09 120 H4H6233P 9.13E+04 1.72E−04 1.88E−09 67H4H6236P 4.20E+04 1.25E−04 2.99E−09 92 H4H6238P 2.41E+04 2.25E−049.32E−09 51 H4H6240P 7.97E+04 3.19E−04 4.00E−09 36 H4H6243P 1.58E+041.78E−04 1.13E−08 65 H4H6245P 7.79E+04 1.17E−04 1.51E−09 98 H4H6246P7.39E+04 1.92E−04 2.59E−09 60 H4H6248P 5.03E+04 7.73E−05 1.54E−09 149H4H6250P 9.01E+04 2.97E−04 3.30E−09 39 H4H6251P 2.82E+04 6.52E−042.31E−08 18 H4H6252P 7.46E+04 7.58E−05 1.02E−09 152 H4H6256P 9.00E+041.07E−04 1.19E−09 108 H4H6260P 9.74E+04 6.51E−05 6.69E−10 177 H4H6269P1.01E+05 6.32E−05 6.25E−10 183 H4H6270P 4.29E+04 1.75E−04 4.08E−09 66H1M2907N 6.20E+04 2.81E−03 4.53E−08 4.1 H2aM2898N NB NB NB NB H2aM2899N8.00E+03 2.00E−03 2.70E−07 5.8 H2aM2901N 1.64E+05 1.45E−03 8.80E−09 8.0H2bM2782N 1.23E+05 1.89E−03 1.53E−08 6.1 H2bM2785N NB NB NB NB H2bM2786N1.80E+05 6.00E−03 3.30E−08 1.9 H2bM2890N 1.00E+04 1.00E−03 1.30E−07 11.6H2bM2891N 2.00E+04 1.20E−03 6.00E−08 9.6 H2bM2906N 8.00E+04 1.30E−031.50E−08 8.9 H3M2788N 8.00E+04 3.50E−04 4.20E−09 33.0 H2bM2783N 2.02E+051.50E−03 7.40E−09 7.7 H3M2929N 1.25E+05 3.62E−03 2.90E−08 3 NB = nobinding under the conditions tested

TABLE 4 Biacore affinities at 37° C. for hGREM1-His binding to capturedanti-GREM1 monoclonal antibodies k_(a) k_(d) K_(D) t_(1/2) mAb captured(1/Ms) (1/s) (M) (min) H4H2895N 1.75E+05 2.15E−04 1.23E−09 54 H4H2780N5.74E+04 1.61E−03 2.80E−08 7 H4H2783N2 1.39E+04 8.49E−04 6.11E−08 14H4H2784N 1.42E+05 1.97E−03 1.38E−08 6 H4H2897N 7.03E+04 2.02E−032.88E−08 6 H4H2889N 1.04E+05 4.22E−04 4.06E−09 27 H4H2892N 1.71E+054.00E−04 2.35E−09 29 H4H2926N 1.26E+05 1.40E−03 1.11E−08 8 H4H6232P1.13E+05 1.95E−04 1.73E−09 59 H4H6233P 1.57E+05 5.12E−04 3.26E−09 23H4H6236P 9.30E+04 2.45E−04 2.63E−09 47 H4H6238P 5.69E+04 9.31E−041.64E−08 12 H4H6240P 1.21E+05 1.18E−03 9.75E−09 10 H4H6243P 7.05E+034.84E−04 6.87E−08 24 H4H6245P 1.03E+05 2.62E−04 2.53E−09 44 H4H6246P1.27E+05 4.44E−04 3.50E−09 26 H4H6248P 8.55E+04 4.87E−04 5.69E−09 24H4H6250P 1.49E+05 8.82E−04 5.93E−09 13 H4H6251P 2.53E+04 6.94E−032.75E−07 2 H4H6252P 1.11E+05 4.59E−04 4.14E−09 25 H4H6256P 1.14E+054.66E−04 4.08E−09 25 H4H6260P 1.66E+05 2.85E−04 1.71E−09 41 H4H6269P1.28E+05 2.54E−04 1.98E−09 46 H4H6270P 9.58E+04 6.50E−04 6.79E−09 18

Example 5. Determination of the GREM1 Inhibitory Activity of theAnti-hGREM1 Antibodies

To further characterize the anti-human Gremlin 1 (GREM1) antibodies,their ability to block GREM1 binding to human bone morphogenetic protein4 (BMP4) was examined via ELISA. Plates were coated with recombinanthuman BMP4 (2 ug/mL) (hBMP4; R&D, #314-BP/CF, residues S293-R408 ofaccession #Q53XC5, expressed in NS0 cells) overnight and then serialdilutions of antibodies were incubated with a constant amount (100 pM)of recombinant human GREM1 protein (hGREM1-His; SEQ ID NO: 595) modifiedwith a biotin tag for 1 hour at 25° C. before this complex was added tocoated plates and allowed to incubate for an additional hour at 25° C.The plates were then washed and plate bound biotin-hGREM1-His wasdetected with streptavidin conjugated with horseradish peroxidase(Pierce, #N200). Plates were then developed with a TMB solution (BDBiosciences, #555214) to produce a colorimetric reaction and thereaction was quenched by acidification with sulfuric acid before readingabsorbance at 450 nm on a PerkinElmer Victor X5 plate reader. Data wereanalyzed using a sigmoidal dose-response model within Prism™ software.The calculated IC₅₀ value, defined as the antibody concentrationrequired to achieve 50% of maximum blocking, was used as an indicator ofblocking potency. The IC₅₀ value for several samples was reported at afixed, lower-bound value of 2.5E-11M, which represents the theoreticallower-limit of this assay, given the fixed concentration ofbiotin-hGREM1-His used in the assay. Percent blockade was calculated asthe ratio of the reduction in signal observed in the presence ofantibody relative to the difference between the signal with GREM1 aloneand background (signal from HRP-conjugated secondary antibody alone).The absorbance measured for the constant concentration of 100 pMbiotin-hGREM1-His alone is defined as 0% blocking and the absorbancemeasured for no added GREM1 is defined as 100% blocking. The absorbancevalues of the wells containing the highest concentration for eachantibody were used to determine the percent maximum blocking. All 24anti-GREM1 antibodies tested in this assay blocked biotin-hGREM1-Hiswith IC₅₀ values ranging from <25 pM to 1.9 nM. At a concentration of 20nM of antibody, the 24 antibodies exhibited from 42 to 96-percentblockade of biotin-hGREM1-His binding to hBMP4.

TABLE 5 Anti-GREM1 antibodies blocking bone morphogenic protein 4(hBMP4) binding to biotin-hGREM1-His IC₅₀ % blocked at maximum Ab PID(M) Ab tested (20 nM) H4H2780N    1.0E−10 69 H4H2783N2  <2.5E−11 76H4H2784N    4.5E−10 59 H4H2889N  <2.5E−11 86 H4H2892N    1.7E−10 82H4H2895N    3.6E−11 94 H4H2897N    4.2E−11 73 H4H2926N    5.2E−11 93H4H6232P  <2.5E−11 93 H4H6233P    6.9E−10 66 H4H6236P  <2.5E−11 95H4H6238P    9.7E−10 64 H4H6240P    6.5E−10 60 H4H6243P    7.2E−12 92H4H6245P    3.6E−11 96 H4H6246P    1.5E−09 62 H4H6248P    2.1E−10 62H4H6250P    1.1E−10 42 H4H6251P    1.1E−10 62 H4H6252P    1.9E−10 53H4H6256P    8.7E−11 75 H4H6260P  <2.5E−11 87 H4H6269P    2.6E−11 73H4H6270P    1.9E−09 62 H4H121N >2.00E−08 5.9 isotype control CalculatedIC50 values <2.50E−11 below theoretical assay bottom and reported as<2.50E−11

Example 6. Effect of GREM1 on BMP4 Signaling

Gremlin 1 (GREM1) is a negative regulator of bone morphogenetic protein(BMP) signaling (Walsh et al. 2010). BMPs belong to the TGF-βsuperfamily and are involved in regulation of many physiologicalprocesses including proliferation, differentiation, and cell-fatedetermination during embryonic and postnatal development (Hogan, 1996).Activation of BMP receptors leads to phosphorylation of SMAD proteinsand transcriptional activation of BMP-responsive genes. GREM1 binds toBMP2, BMP4, and BMP7 and blocks binding to their receptors. A bioassaywas developed to detect the regulation of BMP4 signaling by GREM1 in amammalian cell line, W-20-17, a mouse bone marrow stromal cell linepreviously shown to be responsive to BMP2 (Thies et al. 1992). This cellline was modified to stably express a BMP-responsive luciferasereporter. The resulting stable cell line (W-20-17/BRE-luc cells) wasisolated and maintained in 10% fetal bovine serum, DMEM, NEAA,penicillin/streptomycin, and 200 μg/ml G418.

For the bioassay, the W-20-17/BRE-luc cells were seeded onto 96-wellassay plates at 10,000 cells/well and incubated at 37° C. and 5% CO₂overnight. The next day, recombinant human BMP4 (hBMP4; R&D, #314-BP/CF,residues S293-R408 of accession #Q53XC5, expressed in NS0 cells) wasserially diluted at 1:3 and added to cells starting from 100 nM to 0.002nM including no hBMP4 control for dose response. For inhibition of hBMP4by GREM1, recombinant human GREM1 (hGREM1-His; C-terminal 10His tagged,R&D, #5190-GR, residues K25-D184 of accession #O60565, expressed in NS0cells) was serially diluted at 1:2 starting from 400 nM to 0.4 nMincluding no hGREM1-His control and added to cells along with 200 pM or100 pM hBMP4. For inhibition of hGREM1-His, antibodies were seriallydiluted at 1:3 starting from 100 nM to 0.002 nM including no antibodycontrol and added to cells along with hBMP4 and hGREM1-His at finalconcentrations of either 200 pM and 20 nM or 100 pM and 10 nM,respectively. Luciferase activity was detected after 5.5 hours ofincubation in 37° C. and 5% CO₂.

Thirty-four of the 36 anti-GREM1 antibodies tested in theW-20-17/BRE-luc bioassay fully blocked hGREM1-His inhibition of hBMPsignaling at 10 nM hGREM1-His and 100 pM hBMP4 or 20 nM hGREM1-His and200 pM hBMP4. One antibody, H4H2780N, showed partial blocking ofhGREM1-His and another antibody, H4H6269P, did not inhibit hGREM1-His.Isotype control antibodies (Control mAb1 and Control mAb2) were alsoincluded. IC₅₀ values are shown in Tables 6 and 7. hBMP4 activated theW-20-17/BRE-luc cells with EC₅₀ values of 39 to 116 pM. hGREM1-Hisinhibited 200 pM hBMP4 with an IC₅₀ value of 10.3 nM and 100 pM hBMP4with an IC₅₀ value of 2.9-6.0 nM.

TABLE 6 Inhibition of hGREM-His by anti-human GREM1-antibodies in a cellbased assay hBMP4 EC₅₀ (pM) 116 56 75 73 39 hGrem1-His IC₅₀ 10.3 6.0 4.34.2 5.4 (nM) hBMP4 Constant 200 pM 100 pM hGrem1-His  20 nM  10 nMConstant AbPID IC₅₀ [M] IC₅₀ [M] IC₅₀ [M] IC₅₀ [M] IC₅₀ [M] H2bM2782N3.5E−09 H2bM2785N 2.7E−08 H2bM2786N 1.9E−08 H3M2788N 2.5E−08 H2bM2890N2.1E−09 H2bM2891N 1.1E−09 H2aM2898N 1.4E−09 H2aM2899N 2.1E−09 H2aM2901N1.7E−09 H2bM2906N 1.6E−09 H1M2907N 1.8E−09 H3M2929N 2.2E−09 Control mAb1Not Not Not Not Not Block Block Block Block Block

TABLE 7 Inhibition of hGREM-His by anti-human GREM1-antibodies in a cellbased assay hBMP4 EC₅₀ (pM) 60 hGrem1-His IC₅₀ (nM) 2.9 hBMP4 Constant100 pM hGrem1-His Constant  10 nM AbPID IC₅₀ [M] H4H2780N 49% InhibitionH4H2783N2 1.3E−09 H4H2784N 9.5E−10 H4H2889N 1.2E−09 H4H2892N 9.5E−10H4H2895N 5.8E−10 H4H2897N 4.5E−10 H4H2926N 1.2E−09 H4H6232P 7.8E−10H4H6233P 4.1E−10 H4H6236P 5.4E−10 H4H6238P 1.1E−09 H4H6240P 8.1E−10H4H6243P 5.4E−10 H4H6245P 5.3E−10 H4H6246P 5.6E−10 H4H6248P 5.3E−10H4H6250P 4.4E−10 H4H6251P 6.3E−10 H4H6252P 8.2E−10 H4H6256P 5.4E−10H4H6260P 4.6E−10 H4H6269P Not Block H4H6270P 5.4E−10 Control mAb2 NotBlock

Example 7. Effect of Anti-GREM1 on BMP Signaling and CellDifferentiation

In order to determine the potency of anti-human Gremlin 1 (GREM1)antibodies, their ability to block GREM1 induced inhibition of bonemorphogenetic protein 4 (BMP4) signaling was investigated. W-20-17 cellsare an osteoblast progenitor cell line and can differentiate in responseto BMP4 signaling. GREM1, a known BMP inhibitor, blocks thisdifferentiation. Blocking of GREM1 results in a reversal of BMP4inhibition in this assay. Differentiation can be measuredcolorimetrically by using a substrate to detect endogenous expression ofalkaline phosphatase, an early marker of osteoblast differentiation. Atotal of 24 anti-GREM1 antibodies were tested.

W-20-17 cells were grown in DMEM/10% fetal bovineserum/glutamine/penicillin/streptomycin (complete media) to 100%confluency at 37° C. in 5% CO₂. Cells were washed in 1× PBS, trypsinized(trypsin containing EDTA), plated at 3000 cells/well in clear plastic 96well plates, an\\d grown overnight in complete media at a volume of 100uL/well. The next day recombinant human GREM1 protein (hGREM1-His;C-terminal 10His tagged, R&D, #5190-GR, residues K25-D184 of accession#O60565, expressed in NS0 cells) was mixed with anti-GREM1 antibodies incomplete media and incubated at room temperature (RT) for 40 minutes.Recombinant human BMP4 (hBMP4; R&D, #314-BP/CF, residues S293-R408 ofaccession #Q53XC5, expressed in NS0 cells), also diluted in completemedia, was added to the hGREM1-His/GREM1 antibody mixtures and thenincubated at RT for an additional 30 minutes. After incubation, 50 uL ofthese mixtures was added to W-20-17 cells plated in 100 uL of completemedia. The final concentration of hBMP4 and hGREM1-His on W-20-17 cellsin each well was 1.5 nM and 6 nM, respectively, and the antibodyconcentration varied over an 11-point, 2-fold dilution series (maximumantibody concentration of 200 nM). After 3 days of growth at 37° C. in5% CO₂, media was aspirated and 50 uL of water was added to each well.Ninety-six well plates were frozen at −80° C. for 2 hours and thenthawed on ice. Alkaline phosphatase was measured using p-nitrophenylphosphate substrate prepared as directed (Sigma, #N2770-50SET).Absorbance at 405 nm was measured on a Victor plate reader seven minutesafter addition of 50 uL of substrate. Graphs were plotted in Prism asmean+/−SEM (4 replicates for each condition).

Twenty-two of the 24 anti-GREM1 antibodies tested blocked hGREM1-Hisinhibition of hBMP4 in this assay with IC₅₀ values as shown in Table 8.Two antibodies, H4H6269P and H4H2780N, did not exhibit any measurableblocking of hGREM1-His activity in this assay.

TABLE 8 Gremlin 1 blocking antibodies in W-20-17 cell differentiationassay hGREM1-His Inhibition AbPID IC₅₀, M H4H2895N 1.9E−09 H4H2889N5.4E−09 H4H2892N 4.4E−09 H4H2783N2 5.6E−09 H4H2784N 2.4E−08 H4H2897N1.8E−08 H4H2926N 1.3E−08 H4H6232P 4.5E−09 H4H6233P 2.3E−08 H4H6236P3.9E−09 H4H6238P 2.2E−08 H4H6240P 7.8E−09 H4H6243P 5.9E−09 H4H6245P6.1E−09 H4H6246P 6.2E−09 H4H6248P 5.6E−09 H4H6250P 1.2E−08 H4H6251P1.1E−08 H4H6252P 6.1E−09 H4H6256P 5.7E−09 H4H6260P 3.3E−09 H4H6270P3.1E−09 hBMP4 and hGREM1-His activity in W-20-17 cell differentiationassay: hBMP4 EC₅₀ (M) 5.6E−10 hGREM1-His, IC₅₀ (M) 4.4E−09

Example 8. Generation of a Bi-Specific Antibody

Various bi-specific antibodies are generated for use in practicing themethods of the invention. For example, hGREM1-specific antibodies aregenerated in a bi-specific format (a “bi-specific”) in which variableregions binding to distinct regions of hGREM1 are linked together toconfer dual-domain specificity within a single binding molecule.Appropriately designed bi-specifics may enhance overall GREM1 inhibitoryefficacy through increasing both specificity and binding avidity.Variable regions with specificity for individual regions or epitopes ofGREM1 are paired on a structural scaffold that allows each region tobind simultaneously to the separate epitopes. In one example for abi-specific, heavy chain variable regions (V_(H)) from a binder withspecificity for one region are recombined with light chain variableregions (V_(L)) from a series of binders with specificity for a secondregion to identify non-cognate V_(L) partners that can be paired with anoriginal V_(H) without disrupting the original specificity for thatV_(H). In this way, a single V_(L) segment (e.g., V_(L)1) can becombined with two different V_(H) domains (e.g., V_(H)1 and V_(H)2) togenerate a bi-specific comprised of two binding “arms” (V_(H)1-V_(L)1and V_(H)2-V_(L)1). Use of a single V_(L) segment reduces the complexityof the system and thereby simplifies and increases efficiency incloning, expression, and purification processes used to generate thebi-specific (See, for example, U.S. Ser. No. 13/022,759 andUS2010/0331527).

Alternatively, antibodies that bind to GREM1 and a second target, suchas, but not limited to, for example, a second different anti-GREM1antibody, or a drug specific for fibrosis, may be prepared in abi-specific format using techniques described herein, or othertechniques known to those skilled in the art. Antibody variable regionsbinding to distinct catalytic domain regions may be linked together withvariable regions that bind to relevant sites on, for example, the signalpeptide domain, to confer dual-antigen specificity within a singlebinding molecule. Appropriately designed bi-specifics of this natureserve a dual function. For example, in the case of a bi-specificantibody that binds both the domains, one may be able to betterneutralize both the domains concurrently, without the need foradministration of a composition containing two separate antibodies.Variable regions with specificity for the catalytic domain are combinedwith a variable region with specificity for the signal peptide domainand are paired on a structural scaffold that allows each variable regionto bind to the separate antigens.

Example 9. Inhibition of the GREM1-Heparin Binding Interaction UsingGREM1-Specific Antibodies

In the present study, Bio-Layer Interferometry was used to confirmprevious results showing GREM1 binding to heparin and to evaluate theability of GREM1 specific monoclonal antibodies to interfere with thisbinding interaction. GREM1 and other structurally related cysteineknot-containing proteins including GREM2 and cerberus were also testedfor their ability to bind to heparin. Using these observations, aminoacid residues involved in the binding of GREM1 to heparin werehypothesized by comparing the sequences and known heparin-bindingproperties of other cysteine-knot containing DAN family proteins. Thebinding of GREM1 to heparin was competed by different GAGs includingheparin, HS, and dermatan sulfate (DS) to varying degrees, demonstratingthe specificity of the interaction. Finally, individual antibodies weretested for their ability to interfere with the binding of GREM1 toheparin. Of the twenty-four antibodies tested, four were demonstrated topartially affect aspects of this binding interaction. In an attempt tocompletely block the interaction, the four antibodies that promotedpartial blockade of the GREM1 and heparin binding interaction whentested alone were tested in combination. Some of the combinationsincluding three of the antibodies and the mixture containing all fourantibodies were able to completely inhibit the GREM1 and heparin bindinginteraction. These results give more insight into the binding mechanicsof GREM1 and heparin and demonstrate a possible method usingcombinations of antibodies for inhibiting the angiogenic interaction ofGREM1 with HS for therapeutic treatment.

Reagents and Instrumentation

Carrier free recombinant human GREM1 with a C-terminal decahistidine tagwas obtained from R&D Systems (Minneapolis, Minn.). Heparin-biotinsodium salt from porcine intestinal mucosa and heparin sodium salt fromporcine intestinal mucosa were obtained from Millipore (Billerica,Mass.). Heparan sulfate (HS) sodium salt from porcine intestinal mucosaand dermatan sulfate (DS) sodium salt from porcine intestinal mucosawere obtained from Celsus (Cincinnati, Ohio).

Binding measurements were conducted using an Octet Red96 label-freebiomolecular interaction instrument (ForteBio). All experiments wereperformed at 25° C. using plate agitation at 1000 rpm. Solutions weremade in an aqueous buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA0.05% P20, 0.1 mg/mL BSA and adjusted to pH of 7.4 (HBST buffer).

Binding Kinetics of GREM1 Interacting with Captured Heparin

Super Streptavidin biosensors were loaded with 10 μg/mL heparin-biotinsolutions for 90 seconds. Following a 120 second wash, theheparin-captured biosensors were submerged in wells containing 11.1 nM,33.3 nM, 100 nM, and 300 nM GREM1 solutions to measure association for240 seconds. The biosensors were then submerged in HBST buffer tomeasure dissociation for 240 seconds. The measured association anddissociation rate constants (k_(on)=1.17×10⁶ M⁻¹s⁻¹ andk_(off)=3.27×10⁻³ s⁻¹) demonstrate that GREM1 binds to heparin with highaffinity (K_(D)=k_(off)/k_(on)=2.8×10⁻⁹ M).

Inhibition of GREM1 Binding to Captured Heparin by Glycosaminoglycans

Super Streptavidin biosensors were loaded with 10 μg/mL heparin-biotinsolutions for 90 seconds. Following a 120 second wash, theheparin-captured biosensors were submerged in wells containing 100 nMGREM1 solution pre-mixed with increasing concentrations (0 nM, 20 nM,100 nM, 2 μM) of heparin, heparan sulfate (HS), or dermatan sulfate (DS)to measure association for 240 seconds.

GREM1 exhibited reduced binding to captured heparin-biotin in thepresence of increasing concentrations of heparin, HS, and DS, asreflected in the binding signals observed after 240 seconds ofassociation (Table 9). Exogenously added heparin was most effective inblocking the binding 100 nM GREM1 to captured heparin-biotin, exhibitingcomplete inhibition at 100 nM. Nearly complete inhibition of GREM1binding to captured heparin-biotin was observed when GREM1 was mixedwith 2 μM of HS and DS. The complete inhibition of 100 nM GREM1 bindingto captured heparin-biotin with soluble heparin at 100 nM supports thespecificity of this binding. Partial inhibition with the otherglycosaminoglycans, which are all negatively charged, suggests thatelectrostatic interactions may be important in the GREM1-heparin bindinginteraction.

TABLE 9 The effect of soluble glycosaminoglycans on the GREM1-heparinbinding interaction Binding Response (nm) for 100 nM GREM1 Binding toCaptured Biotin- Heparin in the Presence of Increasing Concentrations ofGlycosaminoglycans Concentration of Heparan Dermatan GlycosaminoglycanHeparin Sulfate Sulfate  0 nM 0.12 0.11 0.09  20 nM 0.07 0.11 0.08 100nM 0 0.07 0.05  2 μM 0 0.04 0.02Inhibition of GREM1 Binding to GREM1 Specific Monoclonal Antibodies byHeparin

Anti-Human IgG Fc Capture biosensors (ForteBio) were separately loadedwith 50 μg/mL solutions of 24 different anti-GREM1 antibodies for 60seconds. Following a 30 second wash, duplicate biosensors for eachantibody were submerged into wells containing either 100 nM GREM1solutions or 100 nM GREM1 solutions containing 5 μM of heparin,measuring association for 300 seconds. The presence or absence ofheparin for some of the antibodies influenced the association rate, andthis effect is summarized in Table 10 where observed signals at both 30seconds and 300 seconds are provided. The binding of antibodiesH4H2895N, H4H2780N, H4H6269P, and H4H2892N to GREM1 was minimallyaffected by the presence of heparin (the binding signals at 30 secondswere reduced by 0.05 nm or less). The binding of eight antibodies(H4H2897N, H4H6252P, H4H6245P, H4H6251P, H4H6232P, H4H2783N2, H4H6236P,and H4H6243P) exhibited the greatest reduction in binding to GREM1 inthe presence of heparin (binding signals reduced by 0.19 nm or greaterat 30 seconds). The other antibodies tested showed intermediate levelsof binding inhibition to GREM1 in the presence of heparin.

TABLE 10 The inhibitory effect of soluble glycosaminoglycans onGREM1-antibody binding interactions 100 nM GREM1 + 100 nM GREM1 5 uMHeparin Binding Binding Binding Binding Reduction Reduction Signal withSignal with Signal with Signal with in Signal in Signal 30 sec 300 sec30 sec 300 sec from from mAb Association Association AssociationAssociation Heparin at Heparin at Captured (nm) (nm) (nm) (nm) 30 sec300 sec H4H2895N 0.15 0.5 0.22 0.54 −0.07 −0.04 H4H2780N 0.13 0.58 0.130.58 0.00 0.00 H4H6269P 0.15 0.6 0.14 0.52 0.01 0.08 H4H2892N 0.2 0.490.15 0.49 0.05 0.00 H4H6233P 0.19 0.47 0.08 0.41 0.11 0.06 H4H6238P 0.130.41 0.02 0.33 0.11 0.08 H4H6246P 0.19 0.38 0.06 0.34 0.13 0.04 H4H6256P0.2 0.4 0.07 0.38 0.13 0.02 H4H6250P 0.2 0.37 0.06 0.32 0.14 0.05H4H2889N 0.2 0.46 0.05 0.44 0.15 0.02 H4H6270P 0.2 0.39 0.05 0.33 0.150.06 H4H2926N 0.23 0.49 0.07 0.42 0.16 0.07 H4H6248P 0.21 0.42 0.04 0.370.17 0.05 H4H6260P 0.24 0.42 0.07 0.37 0.17 0.05 H4H2784N 0.24 0.52 0.060.46 0.18 0.06 H4H6240P 0.23 0.53 0.05 0.37 0.18 0.16 H4H2897N 0.2 0.680.01 0.26 0.19 0.42 H4H6252P 0.23 0.44 0.04 0.35 0.19 0.09 H4H6245P 0.230.41 0.02 0.22 0.21 0.19 H4H6251P 0.27 0.45 0.03 0.36 0.24 0.09 H4H6232P0.33 0.48 0.06 0.46 0.27 0.02 H4H2783N2 0.29 0.47 0.01 0.29 0.28 0.18H4H6236P 0.33 0.45 0.05 0.43 0.28 0.02 H4H6243P 0.3 0.47 0 0.21 0.300.26Inhibition of GREM1 Binding to Heparin by Combinations of GREM1Antibodies

Super Streptavidin biosensors were loaded with 10 μg/mL solutions ofheparin-biotin for 60 seconds. Following a 30 second wash, thebiosensors were submerged into separate GREM1 solutions, each containingall 15 possible combinations of four antibodies (H4H2783N2, H4H2897N,H4H6243P, H4H6245P) chosen from the group in the previous experimentthat exhibited the strongest inhibition of GREM1-heparin binding whentested alone. The mixtures included single antibodies and combinationsof two, three, or four antibodies. Each antibody had a concentration of600 nM in the mixtures. Also included were a negative isotype controlantibody known not to bind to GREM1, two antibodies (H4H2892N andH4H2780N) whose binding to GREM1 was minimally affected by the presenceof heparin, and the reference condition of GREM1 alone. As shown inTable 11, the individual antibodies only partially reduced binding ofGREM1 to captured heparin in this binding format. Combinations ofantibodies decreased binding of GREM1 to captured heparin moreeffectively. The solution containing H4H2897N, H4H6243P, and H4H6245Pand the solution containing all four antibodies completely inhibitedGREM1 from binding to captured heparin.

The GREM1 and heparin binding interaction was completely inhibited usingcombinations of antibodies that were each individually identified to beeffective at partially interfering with this binding interaction. Giventhe highly negatively charged nature of heparin, the results suggestthat heparin binds to GREM1 at multiple positively charged surfaceresidues on GREM1. With this insight, it is proposed that in order tocompletely inhibit the angiogenesis-inducing HS and GREM1 bindinginteraction, multiple antibodies are required that have diverse epitopesoverlapping with multiple heparin-binding sites in the structure ofGREM1.

TABLE 11 The inhibitory effect of GREM1-specific antibodies on theGREM1-heparin binding interaction Binding Response (nm) at 300 sec of100 nM GREM1 binding to captured heparin in the presence of 600 nM ofeach antibody alone PID or as a combination H4H2783N2 0.08 H4H2897N 0.20H4H6243P 0.13 H4H6245P 0.19 H4H2783N2 + H4H2897N 0.06 H4H2783N2 +H4H6243P 0.06 H4H2783N2 + H4H6245P 0.04 H4H2897N + H4H6243P 0.07H4H2897N + H4H6245P 0.19 H4H6243P + H4H6245P 0.05 H4H2783N2 + H4H2897N +0.04 H4H6243P H4H2897N + H4H6243P + −0.01 H4H6245P H4H2783N2 +H4H6243P + 0.02 H4H6245P H4H2783N2 + H4H2897N + 0.10 H4H6245PH4H2783N2 + H4H2897N + −0.01 H4H6243P + H4H6245P H4H2892N 0.40 H4H2780N0.29 no antibody 0.13 negative control antibody 0.12

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A method for treating fibrosis comprisingadministering an effective amount of an antibody or an antigen-bindingfragment thereof to a patient in need thereof, wherein the antibody orantigen-binding fragment binds specifically to human gremlin-1 (GREM1),and comprises the six complementarity determining regions (HCDR1, HCDR2,HCDR3, LCDR1, LCDR2, and LCDR3) contained within the HCVR/LCVR aminoacid sequence pair of SEQ ID NOs: 434/442.
 2. The method of claim 1,wherein the antibody or antigen-binding fragment is administered to thepatient in combination with a second therapeutic agent.
 3. The method ofclaim 1, wherein the antibody or antigen-binding fragment isadministered subcutaneously, intravenously, intradermally, orally, orintramuscularly.
 4. The method of claim 1, wherein the antibody orantigen-binding fragment is administered at a dose of about 0.1 mg/kg ofbody weight to about 100 mg/kg of body weight of the patient.
 5. Themethod of claim 1, wherein the fibrosis is present in liver, lungs orkidney.
 6. The method of claim 1, wherein the fibrosis is selected fromthe group comprising pulmonary fibrosis, pulmonary hypertension,idiopathic pulmonary fibrosis, liver fibrosis, renal fibrosis, diabeticnephropathy, ischemic renal injury, nephrosclerosis and nephrotoxicity.7. The method of claim 1, wherein the antibody or antigen-bindingfragment blocks GREM1 binding to bone morphogenetic protein—2 (BMP2), orBMP4, BMP7 or heparin.
 8. The method of claim 1, wherein the antibody orantigen-binding fragment exhibits one or more properties selected fromthe group consisting of: (a) binds GREM1 at 37° C. with a bindingdissociation equilibrium constant (K_(D)) of less than about 275 nM asmeasured by surface plasmon resonance; (b) binds to GREM1 at 37° C. witha dissociative half-life (t½) of greater than about 3 minutes asmeasured by surface plasmon resonance; (c) binds GREM1 at 25° C. with aKD of less than about 280 nM as measured by surface plasmon resonance;(d) binds to GREM1 at 25° C. with a t½ of greater than about 2 minutesas measured by surface plasmon resonance; and (e) blocks GREM1 bindingto BMP4 with an IC₅₀ of less than about 1.9 nM as measured in acompetition ELISA assay at 25° C.
 9. The method of claim 1, whereinHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise, respectively, theamino acid sequences of SEQ ID NOs: 436-438-440-444-446-448.
 10. Themethod of claim 9, wherein the antibody or antigen-binding fragmentcomprises a HCVR comprising the amino acid sequence of SEQ ID NO: 434,and a LCVR comprising the amino acid sequences of SEQ ID NO:
 442. 11.The method of claim 10, wherein the antibody or antigen-binding fragmentis a full length IgG1 antibody.
 12. The method of claim 10, wherein theantibody or antigen-binding fragment is a full length IgG4 antibody.